Methods of generating libraries and uses thereof

ABSTRACT

This invention relates to methods for the generation of polynucleotide seed libraries and the use of these libraries in generating novel mutants of recombinant proteins and, more particularly, for generating focused libraries of recombinant human antibodies and screening for their affinity binding with target antigens.

CROSS-REFERENCE

This application claims the benefit of U.S. Provisional Application No.60/904,622 (Attorney docket No. 33547-706.101), filed Mar. 1, 2007, U.S.Provisional Application No. 61/020,124 (Attorney docket No.33547-706.102), filed Jan. 9, 2008, U.S. Provisional Application No.60/902,414 (Attorney docket No. 33547-705.101), filed Feb. 20, 2007, andU.S. Provisional Application No. 60/995,970 (Attorney docket no.33547-708.101), filed Sep. 28, 2007, each of which applications isincorporated herein by reference in its entirety.

FIELD OF THE INVENTION

This invention relates to methods for the generation of polynucleotideseed libraries and the use of these libraries in generating novelmutants of recombinant proteins and, more particularly, for generatingfocused libraries of recombinant human antibodies and screening fortheir affinity binding with target antigens.

BACKGROUND OF THE INVENTION

The market for the use of recombinant protein therapeutics has increasedsteadily for the last quarter century. In 2005, six of the top 20 drugswere proteins, and overall, biopharmaceutical drugs accounted forrevenues of approximately $40 billion, of which approximately $17billion was based on the sales of monoclonal antibodies. Monoclonalantibodies represent a distinct class of biotherapeutics with a greatdeal of promise. The antibody scaffold is well tolerated in the clinic,and glycosylated IgG molecules have favorable pharmacokinetic andpharmacodynamic properties. Comparison of the sequences of the approvedantibody drugs, as well as those in development, demonstrates that someof the individual drug molecules are strikingly similar to each other,differing only by a few variations of amino acid residues located in thevariable region of the immunoglobulin.

Typical monoclonal antibodies, like naturally occurring antibodies, havethe appearance of a “Y”-shaped structure and the antigen binding portionbeing located at the end of both short arms of the Y. The typicalantibody molecule consists of four polypeptides—two identical copies ofa heavy (H) chain and two copies of a light (L) chain, forming a generalformula H₂ L2. It is known that each of the heavy chains contains oneN-terminal variable (V_(H)) plus three C-terminal constant (CH₁, CH₂ andCH₃) regions and light chains contain one N-terminal variable (V_(L))and one C-terminal constant (C_(L)) region each. The different variableand constant regions of either heavy or light chains are of roughlyequal length (about 110 amino residues per region). Each light chain islinked to a heavy chain by disulphide bonds and the two heavy chains arelinked to each other by disulphide bonds. Each heavy chain has at oneend a variable domain followed by a number of constant domains, and eachlight chain has a variable domain at one end and a constant domain atthe other end. The light chain variable domain is aligned with thevariable domain of the heavy chain. The light chain constant domain isaligned with the first constant domain of the heavy chain. The remainingconstant domains of the heavy chains are aligned with each other. Theconstant domains in the light and heavy chains are not involved directlyin binding the antibody to the antigen.

Antibodies are typically divided into different classes on the basis ofthe structure of the constant region. In humans for example, five majorstructural classes can be identified immunoglobulin G or IgG, IgM, IgA,IgD and IgE. Each class is distinguished on the basis of its physicaland biological characteristics which relate to the function of theimmunoglobulin in the immune system. IgGs can be further divided intofour subclasses: IgG1, IgG2, IgG3 and IgG4, based on differences in theheavy chain amino acid composition and in disulphide bridging, givingrise to differences in biological behavior. A description of the classesand subclasses is set out in “Essential Immunology” by Ivan Roitt,Blackwell Scientific Publications.

The variable domains of each pair of light and heavy chains form theantigen binding site. They have the same general structure with eachdomain comprising a framework of four regions, whose sequences arerelatively conserved, connected by three complementarity determiningregions (CDRs). The four framework regions (FWs or FRs) largely adopt abeta-sheet conformation and the CDRs form loops connecting, and in somecases comprising part of, the beta-sheet structure. The CDRs are held inclose proximity by the framework regions and, with the CDRs from theother domain, contribute to the formation of the antigen binding site.

The vertebrate immune system has evolved unique genetic mechanisms thatenable it to generate an almost unlimited number of different light andheavy chains in a remarkably economical way by joining separate genesegments together before they are transcribed. The antibody chains areencoded by genes at three separate loci on different chromosomes. Onelocus encodes the heavy chain isotypes and there are separate loci forthe kappa (κ) and lambda (λ) light isotypic chains, although aB-lymphocyte only transcribes from one of these light chain loci. Foreach type of Ig chain—heavy chains, lambda (λ) light chains, and kappa(K) light chain—there is a separate pool of gene segments from which asingle peptide chain is eventually synthesized. Each pool is on adifferent chromosome and usually contains a large number of genesegments encoding the V region of an Ig chain and a smaller number ofgene segments encoding the C region. More specifically, the variableregion of an H-chain comprises three gene fragments, i.e., V, D and Jgene fragments, while the variable region of an L-chain comprises twogene fragments, i.e., J and V gene fragments, regardless of whether theL-chain belongs to a lambda (λ) or kappa (κ) chain. During B celldevelopment a complete coding sequence for each of the two Ig chains tobe synthesized is assembled by site-specific genetic recombination,bringing together the entire coding sequences for a V region and thecoding sequence for a C region.

The large number of inherited V, J and D gene segments available forencoding Ig chains makes a substantial contribution on its own toantibody diversity, but the combinatorial joining of these segmentsgreatly increases this contribution. Further, imprecise joining of genesegments and somatic mutations introduced during the V-D-J segmentjoining at the pre-B cell stage greatly increases the diversity of the Vregions

In addition to these structural characteristics, analyses of naturalantibody sequences together with structural studies have beeninstrumental in revealing how antibodies work (Chothia et al., 1992, J.Mol. Biol., 227: 799-817; Kabat, 1982, Pharmacological Rev., 34: 23-38;Kabat, 1987, Sequences of Proteins of Immunological Interest (NationalInstitutes of Health, Bethesda, Md.)). These studies have shown thatantigen recognition is primarily mediated by complementarity determiningregions (CDRs) that are located at one end of the antibody variabledomain and are connected by β-sheet framework (Wu & Kabat, 1970, J. Exp.Med., 132: 211-250; Kabat & Wu, 1971, Annals New York Acad. Sci., 190:382-393).

The sequence diversity of natural antibodies shows that the CDRs arehypervariable in comparison with the framework, and it is the CDRsequences that determine the antigen specificity of a particularantibody (Jones et al., 1986, Nature, 321: 522-5; Amit et al., 1986,Science, 233: 747-53). These studies have also revealed that the naturalsequence diversity at most CDR positions is not completely random, asbiases for particular amino acids occur in both a site-specific mannerand in terms of overall CDR composition (Davies & Cohen, 1996, Proc.Natl. Acad. Sci. USA, 93: 7-12; Kabat et al., 1977, J. Biol. Chem., 252:6609-16; Zemlin et al., 2003, J. Mol. Biol., 334: 73349; Mian et al.,1991, J. Mol. Biol., 217:133-51; Padlan, 1994, Mol. Immunol., 31:169-217).

In contrast to traditional small molecule based approaches, therapeuticantibodies have significant advantages, including (i) their ability tobe generated and validated quickly; (ii) therapeutic antibodies exhibitfewer side effects and have improved safety profiles, (iii) therapeuticantibodies have well understood pharmacokinetic characteristics, andthey can be optimized to create long half-life products with reduceddosing frequency; iv) therapeutic antibodies are versatile and exhibitflexibility in drug function; v) therapeutic antibody scale-up andmanufacturing processes are robust and well-understood; and vi) theyhave a proven track record of clinical and regulatory success.

Even given the success of monoclonal antibodies, the antibody-as-drugmodality is continuing to evolve, and subject to inefficiency. Further,intrinsic biological bias within the native immune system often worksagainst the more rapid development of improved therapeutics. Theselimitations include, i) the long development time for the isolation ofbiologically active antibodies with affinity constants of therapeuticcaliber, ii) the inability to raise antibodies to certain classes ofprotein targets (intractable targets), and iii) the intrinsic affinityceiling inherent in immune system based affinity selection.

Specifically there is a need for methods to more rapidly developantibodies with improved pharmacokinetics, cross-reactivity, safetyprofiles and superior dosing regimens. Central to this need is thedevelopment of methods that enable the systematic analysis of potentialepitopes with a protein, and enable the selective development ofantibodies with the desired selectivity profiles.

An approach used by a number of companies includes the use of random orsemi random mutagenesis (for example the use of error prone PCR), inconjunction with in vitro molecular evolution. This approach is based onthe creation of random changes in protein structure and the generationof huge libraries of mutant polynucleotides that are subsequentlyscreened for improved variants, usually through the expression of theencoded proteins within a living cell. From these libraries a fewimproved proteins may be selected for further optimization.

Such in vitro mutation approaches are generally limited by the inabilityto systematically search a significant fraction of sequence space, andby the relative difficulty of detecting very rare improvement mutants atheavy mutagenesis loads. This fundamental problem arises because thetotal number of possible mutants for a reasonably sized protein ismassive. For example, a 100 amino acid protein has a potential diversityof 20100 different sequences of amino acids, while existing highthroughput screening methodologies are typically limited to a maximumscreening capacity of 107-108 samples per week. Additionally suchapproaches are relatively inefficient because of redundant codon usage,in which up to around 3¹⁰⁰ of the nucleotide sequences possible for a100 amino acid residue protein actually encode for the same amino acidsand protein, (Gustafsson et al. (2004) Codon Bias and heterologousprotein expression Trends. Biotech. 22 (7) 346-353).

A more sophisticated approach uses a mixture of random mutagenesis withrecombination between protein domains in order to select for improvedproteins (Stemmer Proc. Natl. Acad. Sci. (1994) 91 (22) 10747-51). Thisapproach exploits natural design concepts inherent in protein structuresacross families of proteins, but again requires significant recombinantDNA manipulation and screening capacity of a large number of sequencesto identify rare improvements. Both approaches require extensivefollow-up mutagenesis and analysis to understand the significance ofeach mutation, and to identify the best combination of the manythousands or millions of mutants identified.

SUMMARY OF THE INVENTION

The present invention meets the foregoing and related needs by providingmethods for the generation of polynucleotide libraries, includingsynthetic, semi-synthetic and/or seed libraries, and the use of theselibraries in generating novel mutants of recombinant proteins. Incertain embodiments, the methods provided herein are useful forgenerating focused libraries of recombinant human antibodies andscreening for their affinity binding with target antigens. In oneaspect, a synthetic gene is one that does naturally undergo SHM whenexpressed in a B cell (i.e., an antibody gene). In another aspect, asynthetic gene is one that does not naturally undergo SHM when expressedin a B cell (i.e., a non-antibody gene). In certain embodiments, themethods provided herein are useful for generating focused libraries ofrecombinant non-antibody proteins and screening for enhanced function orreduced susceptibility to somatic hypermutation.

In certain aspects of the present invention, provided herein arecompositions of matter comprising a seed library of polynucleotidesencoding a plurality of one or more polypeptide species of interest thathave at least one region of interest of a protein of interest, whereinthe seed library of polynucleotides comprise at least one syntheticnucleic acid sequence that encodes said at least one region of interestand has been modified to act as a substrate for AID mediated somatichypermutation.

In certain aspects of the present invention, provided herein arecompositions of matter comprising a seed library of polynucleotidesencoding one or more proteins, wherein said seed library ofpolynucleotides comprises at least one synthetic polynucleotide that hasbeen optimized for SHM by insertion of one or more preferred SHM codons.In other aspects, at least one synthetic polynucleotide has beenoptimized for SHM by reducing the density of non-preferred codons.Synthetic polynucleotides can be made resistant to SHM or madesusceptible to SHM using the methods described herein.

In certain aspects, the compositions of the present invention cancomprise a synthetic nucleic acid sequence has been modified to act as asubstrate for AID mediated somatic hypermutation by the insertion ofsomatic hypermutation motifs. In one embodiment, the synthetic nucleicacid sequence has been modified to act as a substrate for AID mediatedsomatic hypermutation by the insertion of one or more preferred SHMcodons. In another embodiment, the synthetic nucleic acid sequence hasbeen modified to act as a substrate for AID mediated somatichypermutation by the insertion of one or more WAC motif, WRC motif or acombination thereof.

In certain other aspects, the compositions of the present inventioncomprise a seed library of polynucleotides encoding a protein ofinterest that is an antibody. In one embodiment, the protein of interestis an antibody heavy chain or fragment thereof. In another embodiment,the antibody heavy chain comprises a variable region selected from thoseset forth in FIG. 20A. In still another embodiment, the antibody heavychain comprises a variable region selected from the group consisting ofIGHV6-1, IGHV4-34, IGHV4-59, IGHV3-30-3, IGHV3-7, IGHV3-23, IGHV5-51,IGHV1-2, or IGHV1-69.

In other embodiments, the protein of interest is an antibody light chainor fragment thereof. In one embodiment, the antibody light chaincomprises a variable region selected from set forth in FIG. 20B. Instill another embodiment, the antibody light chain comprises a κ lightchain variable region selected from the group consisting of IGKV2D-30,IGKV4-1, IGKV1-33, IGKV1D-39, or IGKV3-20. In yet another embodiment,the antibody light chain comprises a variable region selected from setforth in FIG. 20C. In yet still another embodiment, antibody light chaincomprises a λ light chain variable region selected from the groupconsisting of IGKLV7-43, IGLV1-40, IGLV2-11, or IGLV3-21.

In certain embodiments, the compositions of the present inventioncomprise at least one region of interest comprising an antibody heavy orlight chain CDR1, CDR2 or CDR3 domain. In other embodiments, thecompositions comprise at least one said region of interest comprising anantibody heavy or light chain CDR3.

In certain other aspects, the compositions of the present inventioncomprise a protein of interest that is a receptor. In other aspects, theprotein of interest is an enzyme. In still other aspects, the protein ofinterest is a co-factor. In yet other aspects, the protein of interestis a transcription factor.

The present invention also provides a method of making a protein ofinterest with a desired property, the method comprising the steps of: a.synthesizing a seed library of polynucleotides encoding a plurality ofone or more polypeptide species of interest that have at least oneregion of interest of a protein of interest, wherein the seed library ofpolynucleotides comprise at least one synthetic nucleic acid sequencethat encodes at least one region of interest and has been modified toact as a substrate for AID mediated somatic hypermutation; b. joining inoperable combination a seed library of polynucleotides encoding aplurality of one or more polypeptide species of interest of a protein ofinterest into an expression vector; c. transforming a host cell with theexpression vector, so that the protein of interest is produced byexpression of the seed library of polynucleotides encoding a pluralityof one or more polypeptide species of interest of a protein of interest;and wherein the host cell expresses AID, or can be induced to expressAID via the addition of an inducing agent; d. optionally inducing AIDactivity, or allowing AID mediated mutagenesis to occur on the seedlibrary; e. identifying a cell or cells within the population of cellswhich expresses a mutated protein having a desired property, and f.establishing one or more clonal populations of cells from the cell orcells identified in step (e).

In other embodiments, provided herein is a method of making a protein ofinterest with a desired or identified property, said method comprisingthe steps of: (a) synthesizing a seed library of polynucleotidesencoding one or more proteins, wherein said seed library ofpolynucleotides comprises at least one synthetic polynucleotide that hasbeen optimized for SHM; (b) joining in operable combination said seedlibrary of polynucleotides into an expression vector; (c) transforming ahost cell with said expression vector, so that said one or more proteinsis produced by expression of said seed library of polynucleotides; andwherein said host cell expresses AID activity or can be induced toexpress AID activity via the addition of an inducing agent; (d) ifneeded, inducing AID activity; (e) identifying a cell or cells withinthe population of cells which express(es) one or more mutated proteinshaving said desired or identified property, and (f) establishing one ormore clonal populations of cells from the cell or cells identified instep (e).

In other embodiments, provided herein is a method of making an antibodyor antigen-binding fragment thereof with a desired property, the methodcomprising the steps of: a. synthesizing a seed library ofpolynucleotides encoding a plurality of one or more antibody heavy chainproteins or fragments that have at least one CDR, wherein thepolynucleotides comprise at least one synthetic nucleic acid sequencethat encodes the at least one CDR and has been modified to act as asubstrate for AID mediated somatic hypermutation; b. synthesizing a seedlibrary of polynucleotides encoding a plurality of one or more antibodylight chain proteins or fragments that have at least one CDR, whereinthe seed library of polynucleotides comprise at least one syntheticnucleic acid sequence that encodes the at least one CDR and has beenmodified to act as a substrate for AID mediated somatic hypermutation;c. joining in operable combination the seed library of polynucleotidesencoding the plurality of antibody heavy chain proteins or fragmentsthereof and the seed library of polynucleotides encoding the pluralityof antibody light chain proteins or fragments thereof into expressionvectors; d. transforming a host cell with the expression vectors, sothat an antibody or an antigen-binding fragment thereof is produced bycoexpression of a heavy chain sequence from the seed library ofpolynucleotides encoding a plurality of antibody heavy chain proteins orfragments thereof and a light chain sequence from the seed library ofpolynucleotides encoding a plurality of antibody light chain proteins orfragments thereof, either on the same or different expression vectors;and wherein the host cell expresses AID, or can be induced to expressAID via the addition of an inducing agent; e. optionally inducing AIDactivity, or allowing AID mediated mutagenesis to occur on the seedlibraries of polynucleotides; f. identifying a cell or cells within thepopulation of cells which expresses a mutated antibody or anantigen-binding fragment thereof having the desired property, and g.establishing one or more clonal populations of cells from the cell orcells identified in step (f).

In other embodiments, provided herein is a method of making an antibodyor antigen-binding fragment thereof with a desired or identifiedproperty, said method comprising the steps of: (a) synthesizing a firstseed library of first polynucleotides encoding a plurality of one ormore antibody heavy chain proteins or fragments thereof that have atleast one heavy chain CDR, wherein said first seed library ofpolynucleotides comprises at least one first synthetic polynucleotidethat has been optimized for SHM; (b) synthesizing a second seed libraryof second polynucleotides encoding said plurality of one or moreantibody light chain proteins or fragments thereof that have at leastone light chain CDR, wherein said second seed library of polynucleotidescomprises at least one second synthetic polynucleotide that has beenoptimized for SHM; (c) joining in operable combination said first andsecond seed libraries of polynucleotides into expression vectors; (d)transforming a host cell with said expression vectors, so that anantibody or an antigen-binding fragment thereof is produced bycoexpression of a heavy chain sequence from said first seed library ofpolynucleotides and a light chain sequence from said second seed libraryof polynucleotides (either on the same or different expression vectors);and wherein said host cell expresses AID activity or can be induced toexpress AID activity via the addition of an inducing agent; (e) ifneeded, inducing AID activity; (f) identifying a cell or cells withinthe population of cells which expresses one or more mutated antibodiesor antigen-binding fragments thereof having the desired or identifiedproperty, and (g) establishing one or more clonal populations of cellsfrom the cell or cells identified in step (f).

In still other embodiments, provided herein is a method of co-evolving aplurality of proteins, the method comprising the steps of: a.synthesizing a first seed library of polynucleotides encoding aplurality of one or more polypeptide species of interest that have atleast one region of interest of a first protein of interest, wherein theseed library of polynucleotides comprise at least one synthetic nucleicacid sequence that encodes the at least one region of interest and hasbeen modified to act as a substrate for AID mediated somatichypermutation; b. synthesizing a second seed library of polynucleotidesencoding a plurality of one or more polypeptide species of interest thathave at least one region of interest of a second protein of interest,wherein the seed library of polynucleotides comprise at least onesynthetic nucleic acid sequence that encodes the at least one region ofinterest and has been modified to act as a substrate for AID mediatedsomatic hypermutation; c. joining in operable combination the seedlibrary of polynucleotides encoding the plurality of polypeptide speciesof interest of the first protein of interest and the seed library ofpolynucleotides encoding the plurality of polypeptide species ofinterest of the second protein of interest into expression vectors; d.transforming a host cell with the expression vectors, so that the firstand second proteins of interest are produced by coexpression of thefirst and second seed libraries of polynucleotides, either on the sameor different expression vectors; and wherein the host cell expressesAID, or can be induced to express AID via the addition of an inducingagent; e. optionally inducing AID activity, or allowing AID mediatedmutagenesis to occur on the seed libraries of polynucleotides; f.identifying a cell or cells within the population of cells whichexpresses a mutated first or second protein of interest having thedesired property, and g. establishing one or more clonal populations ofcells from the cell or cells identified in step (f).

In one aspect, provided herein is a method of co-evolving a plurality ofproteins, said method comprising the steps of: (a) synthesizing a firstseed library of polynucleotides encoding one or more proteins, whereinsaid first seed library of polynucleotides comprise at least one firstsynthetic polynucleotide that has been optimized for SHM; (b)synthesizing a second seed library of polynucleotides encoding one ormore proteins, wherein said second seed library of polynucleotidescomprise at least one second synthetic polynucleotide that has beenoptimized for SHM; (c) joining in operable combination said first andsecond seed libraries of polynucleotides into expression vectors; (d)transforming a host cell with said expression vectors, so that said oneor more first and second proteins are produced by coexpression of saidfirst and second seed libraries of polynucleotides, either on the sameor different expression vectors; and wherein said host cell expressesAID activity or can be induced to express AID activity via the additionof an inducing agent; (e) if needed, inducing AID activity; (f)identifying a cell or cells within the population of cells whichexpresses one or more mutated proteins having the desired or identifiedproperty, and (g) establishing one or more clonal populations of cellsfrom the cell or cells identified in step (f).

In certain aspects, the methods described herein comprise at least onesynthetic nucleic acid sequence that has been modified to act as asubstrate for AID mediated somatic hypermutation by the insertion ofsomatic hypermutation motifs. In certain embodiments, the at least onesynthetic nucleic acid sequence has been modified to act as a substratefor AID mediated somatic hypermutation by the insertion of one or morepreferred SHM codons. In other embodiments, the at least one syntheticnucleic acid sequence has been modified to act as a substrate for AIDmediated somatic hypermutation by the insertion of one or more WACmotif, WRC motif, or a combination thereof.

In one embodiment of any of these methods, the identified codon may bereplaced with a preferred (canonical) SHM codon or preferred (canonical)hot spot SHM codon which introduces a conservative amino acidsubstitution, compared to either the wild-type or AID modified codon. Inanother embodiment of any of these methods, the identified codon may bereplaced with a preferred SHM codon or preferred hot spot SHM codonwhich introduces a semi-conservative mutation at the amino acid level,compared to either the wild-type or AID modified codon. In anotherembodiment of any of these methods, the identified codon may be replacedwith a preferred SHM codon or preferred hot spot SHM codon whichintroduces a non-conservative mutation at the amino acid level comparedto either the wild-type or AID modified codon. In one embodiment,insertion of one or more preferred SHM codons is by insertion of one ormore amino acids substitutions in said region of interest, said aminoacid substitutions being silent, conservative, semi-conservative,non-conservative or a combination thereof. Modifications topolynucleotides made using the methods described herein can render atleast one polynucleotide sequence susceptible or resistant to SHM.

In certain embodiments, the methods described herein comprise a hostcell that is a prokaryotic cell. In one embodiment, the prokaryotic cellis an E. coli cell.

In certain other embodiments, the methods described herein comprise ahost cell that is a eukaryotic cell. In one embodiment, the eukaryoticcell is a mammalian cell. In another embodiment, the host is a mammaliancell that is a Chinese hamster ovary cell (CHO), a human embryonickidney (HEK) 293 cell, 3T3 cell, a HEK 293T cell, a PER.C6™ cell, or alymphoid derived cell. In still other embodiments, the host cell is alymphoid derived cell that is a RAMOS(CRL-1596) cell, a Daudi (CCL-213)cell, an EB-3 (CCL-85) cell, a DT40 (CRL-2111) cell, an 18-81cell, aRaji (CCL-86), or derivatives thereof.

In another embodiment, the methods described herein comprise a host cellthat is a eukaryotic cell that is a yeast cell.

The present invention further provides a method for humanizing a nonhuman antibody, the method comprising the steps of: a. determining thesequence of the heavy and light chains of the non human antibody to behumanized; b. synthesizing a seed library of polynucleotides encoding aplurality of one or more human antibody heavy chain protein scaffoldscomprising at least one synthetic nucleic acid sequence which encodes atleast one CDR, or a portion thereof, derived from the non human antibodyheavy chain protein, wherein the nucleic acid sequence has been modifiedto act as a substrate for AID mediated somatic hypermutation; c.synthesizing a seed library of polynucleotides encoding a plurality ofone or more human antibody light chain protein scaffolds comprising atleast one synthetic nucleic acid sequence which encodes at least oneCDR, or a portion thereof, derived from the non human antibody lightchain protein, wherein the nucleic acid sequence has been modified toact as a substrate for AID mediated somatic hypermutation; d. joining inoperable combination the seed library of polynucleotides encoding theplurality of antibody heavy chain protein scaffolds and the seed libraryof polynucleotides encoding the plurality of antibody light chainprotein scaffolds into expression vectors; e. transforming a host cellwith the expression vectors, so that an antibody or an antigen-bindingfragment thereof is produced by coexpression of a heavy chain sequencefrom the seed library of polynucleotides encoding the plurality ofantibody heavy chain protein scaffolds and a light chain sequence fromthe seed library of polynucleotides encoding the plurality of antibodylight chain protein scaffolds, either on the same or differentexpression vectors; and wherein the host cell expresses AID, or can beinduced to express AID via the addition of an inducing agent; f.optionally inducing AID activity, or allowing AID mediated mutagenesisto occur on the seed libraries; g. identifying a cell or cells withinthe population of cells which expresses a humanized antibody havingbinding characteristic of the non-human antibody, and h. establishingone or more clonal populations of cells from the cell or cellsidentified in step (g).

In certain embodiments, the method for humanizing a non-human antibodycomprises human antibody heavy chain protein scaffolds comprising avariable region selected from FIG. 20A. In other embodiments, the humanantibody heavy chain protein scaffolds comprise a variable regionselected from FIG. 20A, wherein said selected variable region exhibitsthe highest amino acid homology to said non human antibody. In stillother embodiments, the antibody heavy chain protein scaffolds comprise avariable region selected from the group consisting of IGHV6-1, IGHV4-34,IGHV4-59, IGHV3-30-3, IGHV3-7, IGHV3-23, IGHV5-51, IGHV1-2 or IGHV1-69.

In certain other embodiments, the method for humanizing a non-humanantibody comprises human antibody light chain protein scaffolds comprisea variable region selected from FIG. 20B. In other embodiments, thehuman antibody light chain protein scaffolds comprise a variable regionselected from FIG. 20B, wherein said selected variable region exhibitsthe highest amino acid homology to said non human antibody. In stillother embodiments, the antibody light chain protein scaffolds comprise avariable region selected from the group consisting of IGKV2D-30,IGKV4-1, IGKV1-33, IGKV1D-39, or IGKV3-20.

In certain other embodiments, the method for humanizing a non-humanantibody comprises human antibody light chain protein scaffolds comprisea variable region selected from FIG. 20C. In other embodiments, thehuman antibody light chain protein scaffolds comprise a variable regionselected from FIG. 20C, wherein said selected variable region exhibitsthe highest amino acid homology to said non human antibody. In stillother embodiments, the antibody light chain protein scaffolds comprise avariable region selected from the group consisting of IGKLV7-43,IGLV1-40, IGLV2-11, or IGLV3-21.

In other aspects, the method for humanizing a non-human antibodydescribed herein comprise at least one synthetic nucleic acid sequencehas been modified to act as a substrate for AID mediated somatichypermutation by the insertion of somatic hypermutation motifs. In otheraspects, the at least one synthetic nucleic acid sequence has beenmodified to act as a substrate for AID mediated somatic hypermutation bythe insertion of one or more preferred SHM codons. In still otheraspects, the at least one synthetic nucleic acid sequence has beenmodified to act as a substrate for AID mediated somatic hypermutation bythe insertion of one or more WAC motif, WRC motif, or a combinationthereof.

In other embodiments, the method for humanizing a non-human antibodydescribed herein comprise a plurality of one or more human antibodyheavy chain protein scaffolds comprise a synthetic nucleic acid sequencewhich encodes a CDR3 domain derived from said non human antibody heavychain protein, wherein said nucleic acid sequence has been modified toact as a substrate for AID mediated somatic hypermutation.

In still other embodiments, the method for humanizing a non-humanantibody described herein comprise a plurality of one or more humanantibody light chain protein scaffolds comprise a synthetic nucleic acidsequence which encodes a CDR3 domain derived from said non humanantibody light chain protein, wherein said nucleic acid sequence hasbeen modified to act as a substrate for AID mediated somatichypermutation.

In yet other embodiments, the method for humanizing a non-human antibodydescribed herein comprise a plurality of one or more human antibodyheavy chain protein scaffolds comprise a synthetic nucleic acid sequencewhich encodes a portion of a CDR3 domain derived from said non humanantibody heavy chain protein, wherein said nucleic acid sequence hasbeen modified to act as a substrate for AID mediated somatichypermutation.

In still yet other embodiments, the method for humanizing a non-humanantibody described herein comprise a plurality of one or more humanantibody light chain protein scaffolds comprise a synthetic nucleic acidsequence which encodes a portion of a CDR3 domain derived from said nonhuman antibody light chain protein, wherein said nucleic acid sequencehas been modified to act as a substrate for AID mediated somatichypermutation.

INCORPORATION BY REFERENCE

All publications and patent applications mentioned in this specificationare herein incorporated by reference to the same extent as if eachindividual publication or patent application was specifically andindividually indicated to be incorporated by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

A better understanding of the features and advantages of the presentinvention will be obtained by reference to the following detaileddescription that sets forth illustrative embodiments, in which theprinciples of the invention are utilized, and the accompanying drawingsof which:

FIG. 1 and FIG. 2 show the 20 most common codon transitions, observed inCDRs and FWs during SHM mediated affinity maturation and demonstrate howsimple frame shifts can determine the two radically different patternsof mutagenesis seen in CDRs and FWs. These observations lead directly toa hypothesis that both functional selection during affinity maturationand the reading frame context determines the amino acid diversitygenerated at SHM hot spot codons.

FIG. 1—Shows that within CDRs, (the codons AGC, TAT, and TAC (encodingtyrosine and serine amino acids), feed a directed flow of primary,secondary and tertiary SHM events generating amino acid diversity.Within CDRs, the most common codon transition observed is AGC to AAC(785 instances), leading to a serine to asparagine conversion. Whilethat transitions are also common in framework regions (354 instances), asimple frame shift of the same mutation in the same hotspot motif ( . .. TACAGCTAT . . . ; SEQ ID NO: 1) context leads to a CAG to CAA silentmutation that is common in framework regions (288 instances) but notcommonly observed in CDRs.

FIG. 2—In contrast to FIG. 1, the most commonly observed codon (aminoacid) transition events in frame work regions generate silent mutations(FIG. 2).

FIG. 3—A histogram of all possible 6-mer nucleotide z-scores describingtheir ability to attract (positive z-score) or repel (negative z-score)SHM-mediated mutations. Also shown (at the corresponding z-score) on thedistribution are nucleotide sequences found in the WAC library. Thedotted line indicates the boundary for the top 5% of all SHM recruitinghotspot motifs. As seen in the figure, nucleotide sequences contained inthe WAC library provide a high density of hot spots. The assembly ofdegenerate codons (WACW) results in a subset of possible 4-mer hot spotsdescribed by Rogozin et al. (WRCH), where R=A or G, H=A or C or T, andW=T or A.

FIG. 4—Preferred SHM hot spot codons AAC and TAC, which can be the basisfor a synthetic library, e.g. a seed library, can result in a set ofprimary and secondary mutation events that create considerable aminoacid diversity, as judged by equivalent SHM mutation events observed inIg heavy chains antibodies. From these two codons, basic amino acids(histidine, lysine, arginine), an acidic amino acid (Aspartate),hydrophilic amino acids (serine, threonine, asparagine, tyrosine),hydrophobic amino acids (Alanine, and phenylalanine), and glycine aregenerated as a result of SHM events.

FIG. 5—A histogram of all possible 6-mer nucleotide z-scores describingtheir ability to attract (positive z-score) or repel (negative z-score)SHM-mediated mutations. Also shown (at the corresponding z-score) on thedistribution are nucleotide sequences found in the WRC library. Thedotted line indicates the boundary for the top 5% of all SHM recruitinghotspot motifs. As seen in the figure, nucleotide sequences contained inthe WRC library provide a high density of hot spots. The assembly ofdegenerate codons (WRCW) results in a subset of possible 4-mer hot spotsdescribed by Rogozin et al. (WRCH), where R=A or G, H=A or C or T, andW=T or A.

FIG. 6—The series of mutation events that lead to the creation of aminoacid diversity, starting from “preferred SHM hot spot codons” AGC andTAC, as observed in affinity matured IGV heavy chain sequences. 4200primary and secondary SHM mutation events identified and analyzed fromthe NCBI database, starting from codons encoding asparagine andtyrosine, lead to a set of functionally diverse amino acids.

FIG. 7-Illustrates the convergence of sequence optimization withprogressive iterations of replacement using the program SHMredesign. Thefigure shows both optimization toward an idealized hot and coldsequence, in this case starting with native canine AID nucleotidesequence.

FIG. 8—Provides the amino acid (A; SEQ ID NO: 2), and polynucleotidesequence (B; SEQ ID NO: 3) of native blasticidin gene. Also shown is theinitial analysis of hot spots (C), cold spots (D) and occurrences ofCpGs (E).

FIG. 9—Provides the amino acid (A; SEQ ID NO: 2), and polynucleotidesequence (B; SEQ ID NO: 4) of a synthetic, SHM resistant version of theblasticidin gene. Also shown is the analysis of hot spots (C), coldspots (D) and occurrences of CpGs (E) in the synthetic sequence.

FIG. 10-Provides the amino acid (A; SEQ ID NO: 2), and polynucleotidesequence (B; SEQ ID NO: 5) of a synthetic, SHM susceptible version ofthe blasticidin gene. Also shown is the analysis of hot spots (C), coldspots (D) and occurrences of CpGs (E) in the synthetic sequence.

FIG. 11—Provides a sequence comparison of activation-induced cytidinedeaminase (AID) from Homo sapiens (human; SEQ ID NO: 6), Mus musculus(mouse; SEQ ID NO: 7), Canis familiaris (dog; SEQ ID NO: 8), Rattusnorvegicus (norv-) (rat; SEQ ID NO: 9) and Pan troglodytes (chimpanzee;SEQ ID NO: 10). Variations between the species are represented by boldamino acids.

FIG. 12—Provides the amino acid (A; SEQ ID NO: 11), and polynucleotidesequence (B; SEQ ID NO: 12) of native canine cytidine deaminase (AID)(L198A). Also shown is the analysis of hot spots (C), cold spots (D) andoccurrences of CpGs (E) in the native sequence.

FIG. 13—Provides the polynucleotide sequence (A; SEQ ID NO: 13) of asynthetic SHM susceptible form of canine AID. Also shown is the analysisof hot spots (B), cold spots (C) and occurrences of CpGs (D).

FIG. 14—Provides the polynucleotide sequence (A; SEQ ID NO: 14) of asynthetic SHM resistant form of canine AID. Also shown is the analysisof hot spots (B), cold spots (C) and occurrences of CpGs (D).

FIG. 15—Provides a comparison of cDNA sequences of Canis familiaris(dog; SEQ ID NO: 15) and SHM-optimized (cold) Canis familiaris (dog; SEQID NO: 16), Homo sapiens (human; SEQ ID NO: 17) and Mus musculus (mouse;SEQ ID NO: 18) mRNA activation-induced cytidine deaminase (AID)sequences. GAG sequences are illustrated by bold, underlining.Variations between the sequences are illustrated by bold amino acidresidues.

FIG. 16—Shows the predicted effect of AID activity on reversionfrequency using a protein containing a mutable stop codon such as afluorescent protein (16A). FIG. 16B shows the actual rates of loss offluorescence achieved (shown as GFP extinction) with cells transfectedwith two different concentrations of an expression vector capable ofexpressing AID, and stably expressing GFP. FIG. 16C shows the initialrates of GFP reversion mediated by wild type human AID, and cold canineAID. Also shown is the effect of Ig enhancers on reversion rate.

FIG. 17—Provide schematics of Vector Formats 1 (17A) and 2 (17B).

FIG. 18—Provide schematics of Vector Format 3 (18A) and 4 (18B).

FIG. 19-Provide schematics of Vector Format 5 (19A) and AB184 (19B).

FIG. 20—Shows the frequency with which various immunoglobulin heavyvariable (IgVH) genes are found in the Genbank and PDB databases (20A).FIGS. 20B and 20C provide the same data for the kappa and lambda lightchain variable regions, respectively.

FIG. 21—Illustrates the steps for generating the (A) heavy chain, (B)kappa and (C) lambda light chain libraries.

FIG. 22—Shown is a synthetic CDR3 that contains two circularly permutedideal hot spots (AGCTAC; SEQ ID NO: 19) contained between 2 nonamericideal cold spots (GTCGTCGTC; SEQ ID NO: 20). Here “V” representsvariable domain derived sequences, “D” represents the syntheticpolynucleotide sequence that has been optimized for SHM, but arenaturally derived from CDR3 in the corresponding wild type antibody, “J”represents junction domain derived sequences, and “C” representsconstant domain derived sequences. The synthetic CDR3 is placed withinthe context of the human IGHV4-34, IGHJ1, IgG1 germline sequence as morefully described in Examples 4-7. The nucleotide and amino acid sequencesof FR3, CDR3, FR4 and a portion of the constant region are set forth inSEQ ID NO: 21 and 24, respectively. Alternate CDR3 nucleotide sequencesare set forth as SEQ ID NOS: 22 and 23. Hot spots are underlined and arecontained within 2 nonameric ideal cold spots (italics). Alternate aminoacid sequences are set forth as SEQ ID NOS: 25 and 26.

FIG. 23—Provides a diagram of the synthesis and maturation of Nisin(23A) illustrating amino acid sequences set forth as SEQ ID NOS: 27-30.FIG. 23B-Illustrates a backbone trace of the protein NisC, as describedin the pdb structure 2G0D (www.rcsb.org/pdb/), with residues in thebinding pocket highlighted. A zinc metal and several cysteine, histidineand aspartate residues are the residues that carry out cyclization ofNisA. Here, residues within 10 Angstroms (Å) of the catalytic site arelabeled and shown with a surface representation.

FIG. 24—Provide the polynucleotide sequence of native NisB (SEQ ID NO:31). Also shown is the analysis of hot spots, cold spots and occurrencesof CpGs in the native sequence.

FIG. 25—Provides the polynucleotide sequence of a SHM resistant form ofNisB (SEQ ID NO: 32). Also shown is the analysis of hot spots, coldspots and occurrences of CpGs in the synthetic sequence.

FIG. 26—Provides the polynucleotide sequence of native NisP (SEQ ID NO:33). Also shown is the analysis of hot spots, cold spots and occurrencesof CpGs in the native sequence.

FIG. 27—Provides the polynucleotide sequence of a SHM resistant form ofNisP (SEQ ID NO: 34). Also shown is the analysis of hot spots, coldspots and occurrences of CpGs in the synthetic sequence.

FIG. 28—Provides the polynucleotide sequence of native NisT (28A; SEQ IDNO: 35), and SHM resistant form of NisT (28B; SEQ ID NO: 36).

FIG. 29—Provides the polynucleotide sequence of native NisA (29A; SEQ IDNO: 37), as well as the initial analysis of hot spots (29B), and coldspots (29C). Also shown is a synthetic form of NisA (29D; SEQ ID NO: 38)showing areas of SHM resistant sequence (underlined) and SHM susceptiblesequence, and the analysis of hot (29E) and cold spots (29F).

FIG. 30—Provides the polynucleotide sequence of native NisC (SEQ ID NO:39), as well as the initial analysis of hot spots (30B) and cold spots(30C).

FIG. 31—Shows a synthetic form of NisC (31A; SEQ ID NO: 40) showing theanalysis of hot (31B) and cold spots (31C).

FIG. 32—Provides a schematic of a three zinc-finger protein makingcontacts to a DNA sequence. Each finger is composed of a small betasheet and alpha helix that coordinate a zinc metal ion. While twohistidines and two cysteines bind the zinc, the sidechains of key aminoacids emanate from the beginning of the alpha helix to make basespecific contacts. These positions may be targeted as SHM hotspots wheremutations creating amino acid diversity are desirable. Structural andzinc binding positions of the finger should correspondingly be madecold. ATCGGCGGC (SEQ ID NO:41); TAGCCGCCG (SEQ ID NO: 42).

FIG. 33—Provides a schematic of an individual finger with structurallyconserved positions shown in bold, and residues contacting DNA shownwith a gray background (SEQ ID NO: 43). Portions of the amino acidsequence to be made hot or cold are shown, along with all possiblecorresponding nucleic acid sequences.

V C SEQ ID NO E H SEQ ID NO GTATGC 44 GAACAC 52 GTATGT 45 GAACAT 53GTCTGC 46 GAGCAC 54 GTCTGT 47 GAGCAT 55 GTGTGC 48 GTGTGT 49 GTTTGC 50GTTTGT 51The accompanying z-score for each nucleotide sequence indicates thedegree to which that sequence recruits or repels SHM machinery to thatsite. Individual sequences from these lists may be chosen to enhance orlimit SHM-mediated mutations at each site.

FIG. 34 The 3-mer nucleotide motif AGC represents a preferred site forsomatic hypermutation events. In the Figure, we see the number ofmutations observed in our analysis (line graph) at each position of theAGC motif found in framework (FR) and complementarity-determiningregions (CDR) for the heavy and light chains of antibodies. The fontsize for each nucleotide position of the motif shows how often eachnucleotide serves as the first position of the codon reading frame.Within framework regions, no one reading frame dominates, whereas withinCDRs, the first position (A) of the AGC motif is almost universally usedas the first position of the codon.

FIG. 35 shows the 20 most hot spot codon hypermutation transition eventswithin the FR and CDR regions of heavy chain antibodies, where thenumbers labeling the arrows indicate how often a codon transition eventwas observed. The codons AGC and AGT (serine), and to a lesser extentTAC and TAT (tyrosine), account for ˜50% of the originating mutationsobserved in affinity matured antibodies. Use of these hot spot codonswithin the correct reading frame, combined with affinity maturationleads to many fewer observed silent mutations within CDRs compared toframework regions (highlighted by dotted circles in the figure).

FIG. 36 shows such a transition matrix of transition frequencies for arepresentative SHM system, where the y-axis represents the startingcodon state, and the x-axis represents the ending codon state given around of SHM-mediated evolution. Where off-diagonal gray valuesrepresent possible transitions between codon states, diagonal termsrepresent self-transitions (or non-transitions), and the total matrixhas an off-diagonal normalized evolution rate of 0.5% per generation(selected arbitrarily based-on ideal literature values). Transitionfrequencies are encoded through the use of a grey scale; the actualnumerical values are presented in FIGS. 36B, 36C, 36D and 36E.

FIG. 37 shows the evolution of the codon AGC (serine), a preferred SHMcodon, and the resulting codon frequencies over 50 rounds ofSHM-mediated mutagenesis, as calculated in the Markov chain model.

FIG. 38 shows the evolution of the codon AGC (serine), a preferred SHMcodon, and the resulting amino acid frequencies encoded by the codonsproduced in situ, over 50 rounds of SHM-mediated mutagenesis, ascalculated in the Markov chain model.

FIG. 39 shows the evolution of the codon TCG (serine), a non-preferredSHM codon, and the resulting codon frequencies over 50 rounds ofSHM-mediated mutagenesis, as calculated in the Markov chain model.

FIG. 40 shows the evolution of the codon TCG (serine), a non-preferredSHM codon, and the resulting amino acid frequencies encoded by thecodons produced in situ, over 50 rounds of SHM-mediated mutagenesis, ascalculated in the Markov chain model.

FIG. 41 shows the evolution of the codons AGC/TAC, the “WRC motif”(comprising preferred SHM codons encoding serine and tyrosine) and theresulting codon frequencies over 50 rounds of SHM-mediated mutagenesis,as calculated in the Markov chain model.

FIG. 42 shows the evolution of the codons AGC/TAC, the “WRC motif”(comprising preferred SHM codons encoding serine and tyrosine) and theresulting amino acid frequencies encoded by the codons produced in situ,over 50 rounds of SHM-mediated mutagenesis, as calculated in the Markovchain model.

FIG. 43 shows the evolution of the GGT codon (glycine), a preferred SHMcodon, and the resulting codon frequencies over 50 rounds ofSHM-mediated mutagenesis, as calculated in the Markov chain model. Thefigure shows the immediate evolution of codons arising from singlemutation events, such as GAT (aspartate), GCT (alanine), and AGT(serine). Secondary mutation events acting on these new codons give riseto a tertiary set of codons. For instance, both AGT and GGT under SHMproduce the codon AAT, leading to acquisition of asparagine at thisposition.

FIG. 44 shows the evolution of a GGT codon (glycine), and the immediateevolution of amino acids arising from single mutation events, such asGAT (aspartate), GCT (alanine), and AGT (serine) over 50 rounds ofSHM-mediated mutagenesis, as calculated in the Markov chain model.

FIG. 45 HEK-293 cells transfected with a low affinity anti-HEL antibody(comprising the light chain mutation N31G) and an constitutive AIDexpression vector either after stable transfection and selection (panelsA and C) or transiently with the addition of re-transfected AIDexpression vector (panels B and D) were incubated with either 50 pMHEL-FITC (A and B) or 500 pM HEL-FITC(C and D) and livingHEL-FITC-binding cells were sorted and expanded in culture for anotherround of selection and sequence analysis.

FIG. 46 Previously sorted HEK-293 cells expressing anti-HEL antibodiesand constitutive canine AID either after stable transfection andselection (A and C) or transiently with the addition of re-transfectedAID expression vector (panels B and D) were incubated with either 50 pMHEL-FITC (A and B) or 500 pM HEL-FITC(C and D) and livingHEL-FITC-binding cells were sorted and expanded in culture for anotherround of selection and sequence analysis.

FIG. 47 HEK-293 cells transfected with a low affinity anti-HEL antibodyand evolved over 4 rounds of selection and evolution were analyzed byincubation with 50 pM HEL-FITC, as described in Example 13. Panel Ashows that over 4 rounds of evolution, a clear increase in positivecells is evident in both the FACS scatter plot (panel A), as well astotal number of positive cells gated (panel B).

FIG. 48 Panel A shows a selection of amino sequences around the HyHEL10light chain CDR1 (SEQ ID NOS: 56, 57 and 58), illustrating the evolvedsequence around the site of the Asn 31 mutation introduced in thestarting constructs. Panel B shows the corresponding nucleic acidsequences (SEQ ID NOS: 59, 60 and 61). Panel C shows a representation ofthe measured affinity of the evolved mutants.

FIG. 49. Shows FACS scattergrams for the isolation of antibodies to NGFselected via the use of intact protein over 5 rounds of selection, asdescribed in Example 15. Panels A and B show FACS results using NGFcoupled to beads, and panels C, D and E show FACS scattergrams obtainedusing 50 nM (panel C) or 20 nM (panels D or E) NGF. Inserts to thegraphs show control incubations performed with control cells. In thesegraphs, the X-axis indicates the extent of IgG expression of the cellsand the Y-axis specifies the magnitude of bead binding by cells asdescribed in the Examples.

FIG. 50. Shows the results of Biacore analysis of a representativeantibody isolated from screening of the surface displayed antibodylibrary with NGF as described in Example 15. A multivariate fit of thesedata produce a predicted dissociation constant of (Kd) of 670 nM.

FIG. 51 Provides the polynucleotide sequence (A; SEQ ID NO: 458) of aunmodified form of the Teal Fluorescent Protein (TFP). Also shown is theanalysis of hot spots (B) and cold spots (C) as illustrated by boldcapital letters. 40 CpG methylation sites were present (data not shown).

FIG. 52 Provides the polynucleotide sequence (A; SEQ ID NO: 459) of asynthetic SHM susceptible (hot) form of the Teal Fluorescent Protein(TFP). Also shown is the analysis of hot spots (B) and cold spots (C) asillustrated by bold capital letters. 14 CpG methylation sites werepresent (data not shown).

FIG. 53 Provides the polynucleotide sequence (A; SEQ ID NO: 460) of asynthetic SHM resistant (cold) form of the Teal Fluorescent Protein(TFP). Also shown is the analysis of hot spots (B) and cold spots (C) asillustrated by bold capital letters. 21 CpG methylation sites werepresent (data not shown).

FIG. 53D shows the mutations for a representative segment of the hot andcold TFP constructs. The central row shows the amino acid sequence ofTFP (residues 59 thru 87) in single letter format (SEQ ID NO: 461), andthe “hot” and “cold” starting nucleic acid sequences encoding the twoconstructs are shown above (hot; SEQ ID NO: 462) and below (cold) theamino acid sequence (SEQ ID NO: 463). Mutations observed in the hotsequence are aligned and stacked top of the gene sequences, whilemutations in the cold TFP sequence are shown below. The resultsillustrate how “silent” changes to the coding sequences generatedramatic changes in observed AID-mediated SHM rates, demonstrating thatengineered sequences can be effectively optimized to create fast or slowrates of SHM.

FIG. 53E shows that the spectrum of mutations generated by AID in thepresent in vitro tissue culture system mirror those observed in otherstudies and those seen during in vivo affinity maturation. FIG. 53Eshows the mutations generated in the present study (Box (i) upper left,n=118), and compares them with mutations observed by Zan et al. (box(ii) upper right, n=702), Wilson et al. (lower left, n=25000; box(iii)), and a larger analysis of IGHV chains that have undergoneaffinity maturation (lower right, n=101,926; box (iv)). The Y-axis ineach chart indicates the starting nucleotide, the X-axis indicates theend nucleotide, and the number in each square indicates the percentage(%) of time that nucleotide transition is observed. In the presentstudy, the frequency of mutation transitions and transversions wassimilar to those seen in other data sets. Mutations of C to T and G to Aare the direct result of AID activity on cytidines and account for 48%of all mutation events. In addition, mutations at bases A and T accountfor ˜30% of mutation events (i.e., slightly less than frequenciesobserved in other datasets).

FIG. 53F shows that mutation events are distributed throughout the SHMoptimized nucleotide sequence of the hot TFP gene, with a maximuminstantaneous rate of about 0.08 events per 1000 nucleotides pergeneration centered around 300 nucleotides from the beginning of theopen reading frame. Stable transfection and selection of a gene with AID(for 30 days) produces a maximum rate of mutation of 1 event per 480nucleotides. As a result, genes may contain zero, one, two or moremutations per gene.

FIG. 53G Illustrates the distribution of SHM-mediated events observed inhot TFP sequenced genes compared to the significantly reduced pattern ofmutations seen in cold TFP (FIG. 53H).

DETAILED DESCRIPTION OF THE INVENTION I. Somatic Hypermutation Systems

In vitro somatic hypermutation (SHM) systems as described in relatedpriority application U.S. Provisional Application No. 60/902,414,entitled “SOMATIC HYPERMUTATION SYSTEMS,” filed on Feb. 20, 2007,involve the use of in vitro somatic hypermutation in conjunction withdirected evolution and bioinformatic analysis to create integratedsystems that include, but are not limited to, optimized, controlledsystems for library design, screening, selection and integrated systemsfor the data mining. These systems include:

I. An expression system designed to create SHM susceptible and or SHMresistant DNA sequences, within a cell or cell-free, environment. Thesystem enables the stable maintenance of a mutagenesis system thatprovides for high level targeted SHM in a gene template of interest,while significantly preventing non-specific mutagenesis of structuralproteins, transcriptional control regions and selectable markers.

II. Polynucleotide libraries that are focused in size and specificity.These libraries can be synthetic libraries, semi-synthetic libraries,and/or seed libraries. In certain aspects, the polynucleotide librariescan be enriched for SHM to seed in situ diversity creation. In one suchembodiment, a polynucleotide library can be enriched for SHM wherein thelibrary comprises a plurality of polynucleotides having a nucleic acidsequence encoding a functional portion of a protein of interest that ismodified to act as a substrate for SHM.

III. A process based on computational analysis of protein structure,intra-species and inter-species sequence variation, and the functionalanalysis of protein activity for selecting optimal epitopes that providefor the selection of antibodies with superior selectivity, cross speciesreactivity, and blocking activity.

The overall result of the integration of these approaches is anintegrated system for creating targeted diversity in situ, and for theautomated analysis and selection of proteins with improved traits.

In certain embodiments, the present invention is based in part of animproved understanding of the context of multiple rounds of SHM withinthe reading frame of a polynucleotide sequence, and the underlying logicrelationships inherent within codon usage patterns.

In particular, the above systems for in vitro SHM provide new designpossibilities for the creation of “seed” libraries that can efficientlyserve as the substrate for SHM for the evolution and selection ofimproved proteins.

i. DEFINITIONS

As used herein and in the appended claims, the terms “a,” “an” and “the”can mean, for example, one or more, or at least one, of a unit unlessthe context clearly dictates otherwise. Thus, for example, reference to“an antibody” includes a plurality of such antibodies and reference to“a variable regions” includes reference to one or more variable regionsand equivalents thereof known to those skilled in the art, and so forth.In the event that there is a plurality of definitions for a term herein,those in this section prevail unless stated otherwise.

Where a range of values is provided, it is understood that eachintervening value, to the tenth of the unit of the lower limit unlessthe context clearly dictates otherwise, between the upper and lowerlimits of that range is also specifically disclosed. Each smaller rangebetween any stated value or intervening value in a stated range and anyother stated or intervening value in that stated range is encompassedwithin the invention. The upper and lower limits of these smaller rangesmay independently be included or excluded in the range, and each rangewhere either, neither or both limits are included in the smaller rangesis also encompassed within the invention, subject to any specificallyexcluded limit in the stated range. Where the stated range includes oneor both of the limits, ranges excluding either or both of those includedlimits are also included in the invention.

The terms “comprise” or “comprising” are used in their open,non-limiting sense, that is to say permitting the presence of one ormore features or components in addition to the recited feature orfeatures.

The term “consisting essentially of” refers to a product, particularly apeptide sequence, of a defined number of residues which is notcovalently attached to a larger product. In the case of the peptide ofthe invention referred to above, those of skill in the art willappreciate that minor modifications to the N- or C-terminal of thepeptide may however be contemplated, such as the chemical modificationof the terminal to add a protecting group or the like, e.g. theamidation of the C-terminus.

The term “isolated” refers to the state in which specific bindingmembers or other specific proteins of the invention, or nucleic acidsencoding such binding members or proteins will be, in accordance withthe present invention. Binding members or other proteins, and nucleicacids encoding them will be free or substantially free of material withwhich they are naturally associated such as other polypeptides ornucleic acids with which they are found in their natural environment, orthe environment in which they are prepared (e.g. cell culture) when suchpreparation is by recombinant DNA technology practiced in vitro or invivo. It is to be understood, however, that binding members or otherproteins, and nucleic acids encoding them may be formulated withdiluents or adjuvants and still for practical purposes be isolated—forexample binding members will normally be mixed with gelatin or othercarriers if used to coat microtitre plates for use in immunoassays, orwill be mixed with pharmaceutically acceptable carriers or diluents whenused in diagnosis or therapy. Specific binding members or other specificproteins can be glycosylated, either naturally or by systems ofheterologous eukaryotic cells, or they can be (for example if producedby expression in a prokaryotic cell) unglycosylated.

The term “selection” refers to the separation of one or more members,such as polynucleotides, proteins or cells from a library of suchmembers. Selection can involve both detection and selection, for examplewhere cells are selected by use of a fluorescence activated cell sorter(FACS) that detects a reporter gene and then sorts the cellsaccordingly.

As used herein, “pg” means picogram, “ng” means nanogram, “ug” or “μg”mean microgram, “mg” means milligram, “ul” or “μl” mean microliter, “ml”means milliliter, “1” means liter, “kb” means kilobases, “uM” or “μM”means micromolar, “nM” means nanomolar, “μM” means picomolar, “fM” meansfemtomolar.

The phrase “pharmaceutically acceptable” refers to molecular entitiesand compositions that are physiologically tolerable and do not typicallyproduce an allergic or similar untoward reaction, such as gastric upset,dizziness and the like, when administered to a human.

Antibody Terminology

The term “antibody” describes an immunoglobulin whether natural orpartly or wholly synthetically produced. The term also covers anypolypeptide or protein having a binding domain which is, or ishomologous to, an antigen-binding domain. CDR grafted antibodies arealso contemplated by this term.

“Native antibodies” and “native immunoglobulins” are usuallyheterotetrameric glycoproteins of about 150,000 daltons, composed of twoidentical light (L) chains and two identical heavy (H) chains. Eachlight chain is typically linked to a heavy chain by one covalentdisulfide bond, while the number of disulfide linkages varies among theheavy chains of different immunoglobulin isotypes. Each heavy and lightchain also has regularly spaced intrachain disulfide bridges. Each heavychain has at one end a variable domain (“V_(H)”) followed by a number ofconstant domains (“C_(H)”). Each light chain has a variable domain atone end (“V_(L)”) and a constant domain (“C_(L)”) at its other end; theconstant domain of the light chain is aligned with the first constantdomain of the heavy chain, and the light-chain variable domain isaligned with the variable domain of the heavy chain. Particular aminoacid residues are believed to form an interface between the light- andheavy-chain variable domains.

The term “variable domain” refers to protein domains that differextensively in sequence among family members (i.e. among differentisoforms, or in different species). With respect to antibodies, the term“variable domain” refers to the variable domains of antibodies that areused in the binding and specificity of each particular antibody for itsparticular antigen. However, the variability is not evenly distributedthroughout the variable domains of antibodies. It is concentrated inthree segments called hypervariable regions both in the light chain andthe heavy chain variable domains. The more highly conserved portions ofvariable domains are called the “framework region” or “FR.” The variabledomains of native heavy and light chains each comprise four FRs (FR1,FR2, FR3 and FR4, respectively), largely adopting a β-sheetconfiguration, connected by three hypervariable regions, which formloops connecting, and in some cases forming part of, the β-sheetstructure. The hypervariable regions in each chain are held together inclose proximity by the FRs and, with the hypervariable regions from theother chain, contribute to the formation of the antigen-binding site ofantibodies (see Kabat et al., Sequences of Proteins of ImmunologicalInterest, 5th Ed. Public Health Service, National Institutes of Health,Bethesda, Md. (1991), pages 647-669). The constant domains are notinvolved directly in binding an antibody to an antigen, but exhibitvarious effector functions, such as participation of the antibody inantibody-dependent cellular toxicity.

The term “hypervariable region” when used herein refers to the aminoacid residues of an antibody which are responsible for antigen-binding.The hypervariable region comprises amino acid residues from three“complementarity determining regions” or “CDRs,” which directly bind, ina complementary manner, to an antigen and are known as CDR1, CDR2, andCDR3 respectively.

In the light chain variable domain, the CDRs typically correspond toresidues 24-34 (CDRL1), 50-56 (CDRL2) and 89-97 (CDRL3), and in theheavy chain variable domain the CDRs typically correspond to residues31-35 (CDRH1), 50-65 (CDRH2) and 95-102 (CDRH3); Kabat et al., Sequencesof Proteins of Immunological Interest, 5th Ed. Public Health Service,National Institutes of Health, Bethesda, Md. (1991)) and/or thoseresidues from a “hypervariable loop” (i.e. residues 26-32 (L1), 50-52(L2) and 91-96 (L3) in the light chain variable domain and 26-32 (H1),53-55 (H2) and 96-101 (H3) in the heavy chain variable domain; Chothiaand Lesk J. Mol. Biol. 196:901 917 (1987)).

As used herein, “variable framework region” or “VFR” refers to frameworkresidues that form a part of the antigen binding pocket or groove and/orthat may contact antigen. In some embodiments, the framework residuesform a loop that is a part of the antigen binding pocket or groove. Theamino acids residues in the loop may or may not contact the antigen. Inan embodiment, the loop amino acids of a VFR are determined byinspection of the three-dimensional structure of an antibody, antibodyheavy chain, or antibody light chain. The three-dimensional structuremay be analyzed for solvent accessible amino acid positions as suchpositions are likely to form a loop and/or provide antigen contact in anantibody variable domain. Some of the solvent accessible positions cantolerate amino acid sequence diversity and others (e.g. structuralpositions) will be less diversified. The three dimensional structure ofthe antibody variable domain may be derived from a crystal structure orprotein modeling. In some embodiments, the VFR comprises, consistessentially of, or consists of amino acid positions corresponding toamino acid positions 71 to 78 of the heavy chain variable domain, thepositions defined according to Kabat et al., 1991. In some embodiments,VFR forms a portion of Framework Region 3 located between CDRH2 andCDRH3. Preferably, VFR forms a loop that is well positioned to makecontact with a target antigen or form a part of the antigen bindingpocket.

Depending on the amino acid sequence of the constant domain of theirheavy chains, immunoglobulins can be assigned to different classes.There are five major classes of immunoglobulins: IgA, IgD, IgE, IgG, andIgM, and several of these may be further divided into subclasses(isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2. The heavy-chainconstant domains (Fc) that correspond to the different classes ofimmunoglobulins are called α, δ, ε, γ, and μ, respectively. The subunitstructures and three-dimensional configurations of different classes ofimmunoglobulins are well known.

The “light chains” of antibodies (immunoglobulins) from any vertebratespecies can be assigned to one of two clearly distinct types, calledkappa or (“κ”) and lambda or (“λ”), based on the amino acid sequences oftheir constant domains.

The terms “antigen-binding portion of an antibody,” “antigen-bindingfragment,” “antigen-binding domain,” “antibody fragment” or a“functional fragment of an antibody” are used interchangeably in thepresent invention to mean one or more fragments of an antibody thatretain the ability to specifically bind to an antigen, (see generally,Holliger et al., Nature Biotech. 23 (9) 1126-1129 (2005)). Non-limitingexamples of antibody fragments included within, but not limited to, theterm “antigen-binding portion” of an antibody include (i) a Fabfragment, a monovalent fragment consisting of the V_(L), V_(H), C_(L)and C_(H1) domains; (ii) a F(ab′)₂ fragment, a bivalent fragmentcomprising two Fab fragments linked by a disulfide bridge at the hingeregion; (iii) a Fd fragment consisting of the V_(H) and C_(H1) domains;(iv) a Fv fragment consisting of the V_(L) and V_(H) domains of a singlearm of an antibody, (v) a dAb fragment (Ward et al., (1989) Nature341:544 546), which consists of a V_(H) domain; and (vi) an isolatedcomplementarity determining region (CDR). Furthermore, although the twodomains of the Fv fragment, V_(L) and V_(H), are coded for by separategenes, they can be joined, using recombinant methods, by a syntheticlinker that enables them to be made as a single protein chain in whichthe V_(L) and V_(H) regions pair to form monovalent molecules (known assingle chain Fv (scFv); see e.g., Bird et al. (1988) Science 242:423426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA 85:5879 5883;and Osbourn et al. (1998) Nat. Biotechnol. 16:778). Such single chainantibodies are also intended to be encompassed within the term“antigen-binding portion” of an antibody. Any V_(H) and V_(L) sequencesof specific scFv can be linked to human immunoglobulin constant regioncDNA or genomic sequences, in order to generate expression vectorsencoding complete IgG molecules or other isotypes. V_(H) and V_(L) canalso be used in the generation of Fab, Fv or other fragments ofimmunoglobulins using either protein chemistry or recombinant DNAtechnology. Other forms of single chain antibodies, such as diabodiesare also encompassed.

“F(ab′)₂” and “Fab” moieties can be produced by treating immunoglobulin(monoclonal antibody) with a protease such as pepsin and papain, andincludes an antibody fragment generated by digesting immunoglobulin nearthe disulfide bonds existing between the hinge regions in each of thetwo H chains. For example, papain cleaves IgG upstream of the disulfidebonds existing between the hinge regions in each of the two H chains to.generate two homologous antibody fragments in which an L chain composedof V_(L) (L chain variable region) and C_(L) (L chain constant region),and an H chain fragment composed of V_(H)(H chain variable region) andC_(Hγ1) (γ₁ region in the constant region of H chain) are connected attheir C terminal regions through a disulfide bond. Each of these twohomologous antibody fragments is called Fab′. Pepsin also cleaves IgGdownstream of the disulfide bonds existing between the hinge regions ineach of the two H chains to generate an antibody fragment slightlylarger than the fragment in which the two above-mentioned Fab′ areconnected at the hinge region. This antibody. fragment is calledF(ab′)₂.

The Fab fragment also contains the constant domain of the light chainand the first constant domain (C_(H1)) of the heavy chain. Fab′fragments differ from Fab fragments by the addition of a few residues atthe carboxyl terminus of the heavy chain C_(H1) domain including one ormore cysteine(s) from the antibody hinge region. Fab′-SH is thedesignation herein for Fab′ in which the cysteine residue(s) of theconstant domains bear a free thiol group. F(ab′)₂ antibody fragmentsoriginally were produced as pairs of Fab′ fragments which have hingecysteines between them. Other chemical couplings of antibody fragmentsare also known.

“Fv” is the minimum antibody fragment which contains a completeantigen-recognition and antigen-binding site. This region consists of adimer of one heavy chain and one light chain variable domain in tight,non-covalent association. It is in this configuration that the threehypervariable regions of each variable domain interact to define anantigen-binding site on the surface of the V_(H)-V_(L) dimer.Collectively, the six hypervariable regions confer antigen-bindingspecificity to the antibody. However, even a single variable domain (orhalf of an Fv comprising only three hypervariable regions specific foran antigen) has the ability to recognize and bind antigen, although at alower affinity than the entire binding site.

“Single-chain Fv” or “sFv” antibody fragments comprise the V_(H) andV_(L) domains of an antibody, wherein these domains are present in asingle polypeptide chain. Generally, the Fv polypeptide furthercomprises a polypeptide linker between the V_(H) and V_(L) domains whichenables the sFv to form the desired structure for antigen binding. For areview of sFv see Pluckthun in The Pharmacology of MonoclonalAntibodies, vol. 113, Rosenburg and Moore eds. Springer-Verlag, NewYork, pp. 269 315 (1994).

The term “Avimer™” refers to a new class of therapeutic proteins thatare from human origin, which are unrelated to antibodies and antibodyfragments, and are composed of several modular and reusable bindingdomains, referred to as A-domains (also referred to as class A module,complement type repeat, or LDL-receptor class A domain). They weredeveloped from human extracellular receptor domains by in vitro exonshuffling and phage display, (Silverman et al., 2005, Nat. Biotechnol.23:1493-94; Silverman et al., 2006, Nat. Biotechnol. 24:220). Theresulting proteins may comprise multiple independent binding domainsthat may exhibit improved affinity (in some cases sub-nanomolar) andspecificity compared with single-epitope binding proteins. See, forexample, U.S. Patent Application Publ. Nos. 2005/0221384, 2005/0164301,2005/0053973 and 2005/0089932, 2005/0048512, and 2004/0175756, each ofwhich is hereby incorporated by reference herein in its entirety.

Each of the known 217 human A-domains comprises ˜35 amino acids (˜4 kDa)and domains are separated by linkers that average five amino acids inlength. Native A-domains fold quickly and efficiently to a uniform,stable structure mediated primarily by calcium binding and disulfideformation. A conserved scaffold motif of only 12 amino acids is requiredfor this common structure. The end result is a single protein chaincontaining multiple domains, each of which represents a separatefunction. Each domain of the proteins binds independently and that theenergetic contributions of each domain are additive. These proteins werecalled “Avimersm” from avidity multimers.

As used herein, “natural” or “naturally occurring” antibodies orantibody variable domains, refers to antibodies or antibody variabledomains having a sequence of an antibody or antibody variable domainidentified from a nonsynthetic source, for example, from adifferentiated antigen-specific B cell obtained ex vivo, or itscorresponding hybridoma cell line, or from the serum of an animal. Theseantibodies can include antibodies generated in any type of immuneresponse, either natural or otherwise induced. Natural antibodiesinclude the amino acid sequences, and the nucleotide sequences thatconstitute or encode these antibodies, for example, as identified in theKabat database.

The terms “synthetic polynucleotide,” “synthetic gene” or “syntheticpolypeptide,” as used herein, mean that the corresponding polynucleotidesequence or portion thereof, or amino acid sequence or portion thereof,is derived, from a sequence that has been designed, or synthesizedde-novo, or modified, compared to the equivalent naturally occurringsequence. Synthetic polynucleotides or synthetic genes can be preparedby methods known in the art, including but not limited to, the chemicalsynthesis of nucleic acid or amino acid sequences or amplified via PCR(or similar enzymatic amplification systems). Synthetic genes aretypically different from unmodified genes or naturally occurring genes,either at the amino acid, or polynucleotide level (or both) and are,typically, located within the context of synthetic expression controlsequences. For example, synthetic gene sequences may include amino acid,or polynucleotide, sequences that have been changed, for example, by thereplacement, deletion, or addition, of one or more, amino acids, ornucleotides, thereby providing an antibody amino acid sequence, or apolynucleotide coding sequence that is different from the sourcesequence. Synthetic gene or polynucleotide sequences may not necessarilyencode proteins with different amino acids, compared to the naturalgene, for example, they can also encompass synthetic polynucleotidesequences that incorporate different codons but which encode the sameamino acid; i.e. the nucleotide changes represent silent mutations atthe amino acid level. In one embodiment, synthetic genes exhibit alteredsusceptibility to SHM compared to the naturally occurring or unmodifiedgene. Synthetic genes can be iteratively modified using the methodsdescribed herein and, in each successive iteration, a correspondingpolynucleotide sequence or amino acid sequence, is derived, in whole orpart, from a sequence that has been designed, or synthesized de novo, ormodified, compared to an equivalent unmodified sequence.

The terms “semi-synthetic polynucletide” or “semi-synthetic gene,” asused herein, refer to polynucleotide sequences that consist in part of anucleic acid sequence that has been obtained via polymerase chainreaction (PCR) or other similar enzymatic amplification system whichutilizes a natural donor (i.e., peripheral blood monocytes) as thestarting material for the amplification reaction. The remaining“synthetic” polynucleotides, i.e., those portions of semi-syntheticpolynucleotide not obtained via PCR or other similar enzymaticamplification system can be synthesized de novo using methods known inthe art including, but not limited to, the chemical synthesis of nucleicacid sequences.

The term “synthetic variable regions” refers to synthetic polynucleotidesequences that are substantially comprised of optimal SHM hot spots andhot codons that, when combined with the activity of AID and/or one ormore error-prone polymerases, can generate a broad spectrum of potentialamino acid diversity at each position. Synthetic variable regions may beseparated by synthetic frame work sequences that encompass codons thatare not specifically targeted for SHM, or that are resistant to SHM butthat provide an optimal context for mutagenesis.

The term “diabodies” refers to small antibody fragments with twoantigen-binding sites, which fragments comprise a heavy chain variabledomain (V_(H)) connected to a light chain variable domain (V_(L)) in thesame polypeptide chain (V_(H)-V_(L)). By using a linker that is tooshort to allow pairing between the two domains on the same chain, thedomains are forced to pair with the complementary domains of anotherchain and create two antigen-binding sites. Diabodies are described morefully in, for example, EP 404,097; WO 93/11161; and Hollinger et al.,Proc. Natl. Acad. Sci. USA 90:6444 6448 (1993).

Antibodies of the present invention also include heavy chain dimers,such as antibodies from camelids and sharks. Camelid and sharkantibodies comprise a homodimeric pair of two chains of V-like andC-like domains (neither has a light chain). Since the V_(H) region of aheavy chain dimer IgG in a camelid does not have to make hydrophobicinteractions with a light chain, the region in the heavy chain thatnormally contacts a light chain is changed to hydrophilic amino acidresidues in a camelid. V_(H) domains of heavy-chain dimer IgGs arecalled V_(HH) domains. Shark Ig-NARs comprise a homodimer of onevariable domain (termed a V-NAR domain) and five C-like constant domains(C-NAR domains).

In camelids, the diversity of antibody repertoire is determined by thecomplementary determining regions (CDR) 1, 2, and 3 in the V_(H) orV_(HH) regions. The CDR3 in the camel V_(HH) region is characterized byits relatively long length averaging 16 amino acids (Muyldermans et al.,1994, Protein Engineering 7(9): 1129). This is in contrast to CDR3regions of antibodies of many other species. For example, the CDR3 ofmouse V_(H) has an average of 9 amino acids.

Libraries of camelid-derived antibody variable regions, which maintainthe in vivo diversity of the variable regions of a camelid, can be madeby, for example, the methods disclosed in U.S. Patent Application Ser.No. 20050037421, published Feb. 17, 2005.

“Humanized” forms of non-human (e.g., murine) antibodies are chimericantibodies which contain minimal sequence derived from non-humanimmunoglobulin. For the most part, humanized antibodies are humanimmunoglobulins (recipient antibody) in which hypervariable regionresidues of the recipient are replaced by hypervariable region residuesfrom a non-human species (donor antibody) such as mouse, rat, rabbit ornonhuman primate having the desired specificity, affinity, and capacity.In some instances, framework region (FR) residues of the humanimmunoglobulin are replaced by corresponding non-human residues.Furthermore, humanized antibodies may comprise residues which are notfound in the recipient antibody or in the donor antibody. Thesemodifications are made to further refine antibody performance. Ingeneral, the humanized antibody will comprise substantially all of atleast one, and typically two, variable domains, in which all orsubstantially all of the hypervariable regions correspond to those of anon-human immunoglobulin and all, or substantially all, of the FRs arethose of a human immunoglobulin sequence. The humanized antibodyoptionally also will comprise at least a portion of an immunoglobulinconstant region (Fc), typically that of a human immunoglobulin. Forfurther details, see Jones et al., Nature 321:522 525 (1986); Reichmannet al., Nature 332:323 329 (1988); and Presta, Curr. Op. Struct. Biol.2:593 596 (1992).

A “humanized antibody” of the present invention includes synthetic andsemi-synthetic antibodies prepared by in vitro somatic hypermutationdriven affinity maturation of library-derived polynucleotides.Specifically included are monoclonal antibodies in which part, or all ofthe complementarity determining regions of the heavy and light chain arederived from a non-human monoclonal antibody, substantially all theremaining portions of the variable regions are derived from humanvariable region templates as described herein (both heavy and lightchain), and the constant regions are derived from human constant regiontemplates likewise described herein. In one aspect, such non-human CDRsequences comprise synthetic polynucleotide sequences that have beenoptimized for somatic hypernutation, and comprise preferred SHM codons,e.g., preferred SHM hot spot codons. In one embodiment, the CDR3 regionsof the heavy and light chain are derived from the non-human antibody.

The term “monoclonal antibody” as used herein refers to an antibodyobtained from a population of substantially homogeneous antibodies,i.e., the individual antibodies comprising the population are identicalexcept for possible naturally occurring mutations that may be present inminor amounts. Monoclonal antibodies are highly specific, being directedagainst a single antigenic site. Furthermore, in contrast toconventional (polyclonal) antibody preparations which include differentantibodies directed against different determinants (epitopes), eachmonoclonal antibody is directed against a single determinant on theantigen. The modifier “monoclonal” indicates the character of theantibody as being obtained from a substantially homogeneous populationof antibodies, and is not to be construed as requiring production of theantibody by any particular method. For example, the monoclonalantibodies to be used in accordance with, the present invention may bemade by the hybridoma method first described by Kohler et al., Nature256:495 (1975), or may be made by recombinant DNA methods (see, e.g.,U.S. Pat. No. 4,816,567). In certain embodiments, the “monoclonalantibodies” may also be isolated from phage antibody libraries using thetechniques described in Clackson et al., Nature 352:624 628 (1991) andMarks et al., J. Mol. Biol. 222:581 597 (1991), for example.

In other embodiments, monoclonal antibodies can be isolated and purifiedfrom the culture supernatant or ascites mentioned above by saturatedammonium sulfate precipitation, euglobulin precipitation method, caproicacid method, caprylic acid method, ion exchange chromatography (DEAE orDE52), affinity chromatography using anti-immunoglobulin column orprotein A column.

A polyclonal antibody (antiserum) or monoclonal antibody of the presentinvention can be produced by known methods. Namely, mammals, preferably,mice, rats, hamsters, guinea pigs, rabbits, cats, dogs, pigs, goats,horses, or cows, or more preferably, mice, rats, hamsters, guinea pigs,or rabbits are immunized, for example, with an antigen mentioned abovewith Freund's adjuvant, if necessary. The polyclonal antibody can beobtained from the serum obtained from the animal so immunized. Themonoclonal antibodies are produced as follows. Hybridomas are producedby fusing the antibody-producing cells obtained from the animal soimmunized and myeloma cells incapable of producing autoantibodies. Thenthe hybridomas are cloned, and clones producing the monoclonalantibodies showing the specific affinity to the antigen used forimmunizing the mammal are screened.

An “isolated specific binding member” is one which has been identifiedand separated and/or recovered from a component of its naturalenvironment. Contaminant components of its natural environment arematerials which would interfere with diagnostic or therapeutic uses forthe specific binding member, and may include enzymes, hormones, andother proteinaceous or nonproteinaceous solutes. In preferredembodiments, the specific binding member will be purified (1) to greaterthan 95% by weight as determined by the Lowry or comparable assaymethod, and most preferably more than 99% by weight, (2) to a degreesufficient to obtain at least 15 residues of N-terminal or internalamino acid sequence by use of a spinning cup sequenator, or (3) tohomogeneity by SDS-PAGE under reducing or nonreducing conditions usingCoomassie blue or, preferably, silver stain. Isolated specific bindingmembers include those in situ within recombinant cells since at leastone component of the specific binding member's natural environment willnot be present. Ordinarily, however, isolated specific binding memberswill be prepared by at least one purification step.

As used herein, an “intrabody or fragment thereof” refers to antibodiesthat are expressed and function intracellularly. Intrabodies typicallylack disulfide bonds and are capable of modulating the expression oractivity of target genes through their specific binding activity.Intrabodies include single domain fragments such as isolated V_(H) andV_(L) domains and scFvs. An intrabody can include sub-cellulartrafficking signals attached to the N or C terminus of the intrabodiesto allow them to be expressed at high concentrations in the sub-cellularcompartments where a target protein is located. Upon interaction withthe target gene, an intrabody modulates target protein function, and/orachieves phenotypic/functional knockout by mechanisms such asaccelerating target protein degradation and sequestering the targetprotein in a non-physiological sub-cellular compartment. Othermechanisms of intrabody-mediated gene inactivation can depend on theepitope to which the intrabody is directed, such as binding to thecatalytic site on a target protein or to epitopes that are involved inprotein-protein, protein-DNA or protein-RNA interactions. In oneembodiment, an intrabody is a scFv.

The “cell producing an antibody reactive to a protein or a fragmentthereof” of the present invention means any cell producing theabove-described antibodies or. antigen-binding fragments of the presentinvention.

The term “germline gene segments” refers to the genes from the germline(the haploid gametes and those diploid cells from which they areformed). The germline DNA contain multiple gene segments that encode asingle immunoglobin heavy or light chain. These gene segments arecarried in the germ cells but cannot be transcribed and translated intoheavy and light chains until they are arranged into functional genes.During B-cell differentiation in the bone marrow, these gene segmentsare randomly shuffled by a dynamic genetic system capable of generatingmore than 108 specificities. Most of these gene segments are publishedand collected by the germline database.

As used herein, “library” refers to a plurality of polynucleotides,proteins, or cells comprising a collection of two, or two or more,non-identical but related members. A “synthetic library” refers to aplurality of synthetic polynucleotides, or a population of cells thatcomprise said plurality of synthetic polynucleotides. A “semi-syntheticlibrary” refers to a plurality of semi-synthetic polynucleotides, or apopulation of cells that comprise said plurality of semi-syntheticpolynucleotides. A “seed library” refers to a plurality of one or moresynthetic or semi-synthetic polynucleotides, or cells that comprise saidpolynucleotides, that contain one or more sequences or portions thereof,that have been modified to act as a substrate for SHM, e.g.,AID-mediated somatic hypermutatin, and that are capable, when acted uponby somatic hypermutation, to create a library of polynucleotides,proteins or cells in situ.

“Antigen” refers to substances that are capable, under appropriateconditions, of inducing a specific immune response and of reacting withthe products of that response, that is, with specific antibodies orspecifically sensitized T-lymphocytes, or both. Antigens may be solublesubstances, such as toxins and foreign proteins, or particulates, suchas bacteria and tissue cells; however, only the portion of the proteinor polysaccharide molecule known as the antigenic determinant (epitopes)combines with the antibody or a specific receptor on a lymphocyte. Morebroadly, the term “antigen” may be used to refer to any substance towhich an antibody binds, or for which antibodies are desired, regardlessof whether the substance is immunogenic. For such antigens, antibodiesmay be identified by recombinant methods, independently of any immuneresponse.

The term “affinity” refers to the equilibrium constant for thereversible binding of two agents and is expressed as Kd. Affinity of abinding protein to a ligand such as affinity of an antibody for anepitope can be, for example, from about 100 nanomolar (nM) to about 0.1nM, from about 100 nM to about 1 picomolar (pM), or from about 100 nM toabout 1 femtomolar (fM). The term “avidity” refers to the resistance ofa complex of two or more agents to dissociation after dilution.

“Epitope” refers to that portion of an antigen or other macromoleculecapable of forming a binding interaction with the variable regionbinding pocket of an antibody. An epitope can be a linear peptidesequence (i.e., “continuous”) or can be composed of noncontiguous aminoacid sequences (i.e., “conformational” or “discontinuous”). An antibodyor antigen-binding fragment can recognize one or more amino acidsequences; therefore an epitope can define more than one distinct aminoacid sequence. Epitopes recognized by an antibody or antigen-bindingfragment can be determined by peptide mapping and sequence analysistechniques well known to one of skill in the art. Typically, suchbinding interaction is manifested as an intermolecular contact with oneor more amino acid residues of a CDR. Often, the antigen bindinginvolves a CDR3 or a CDR3 pair.

A “cryptic epitope” or a “cryptic binding site” is an epitope or bindingsite of a protein sequence that is not exposed or substantiallyprotected from recognition within at least one native conformation ofthe polypeptide, but is capable of being recognized by an antibody orantigen-binding fragment in a second conformation of the polypeptide, orin the denatured, or proteolyzed polypeptide. Amino acid sequences thatare not exposed, or are only partially exposed, in only one specificnative conformation of the polypeptide structure are potential crypticepitopes. If an epitope is not exposed, or only partially exposed, thenit is likely that it is buried within the interior of the polypeptide,or masked by an interaction with a macromolecular structure. Candidatecryptic epitopes can be identified, for example, by examining thethree-dimensional structure of a native polypeptide.

The term “specific” may be used to refer to the situation in which onemember of a specific binding pair will not show any significant bindingto molecules other than its specific binding partner(s). The term isalso applicable where e.g. an antigen binding domain is specific for aparticular epitope which is carried by a number of antigens, in whichcase the specific binding member carrying the antigen binding domainwill be able to bind to the various antigens carrying the epitope.

The term “binding” refers to a direct association between two molecules,due to, for example, covalent, electrostatic, hydrophobic, and ionicand/or hydrogen-bond interactions under physiological conditions, andincludes interactions such as salt bridges and water bridges.

The term “specific binding member” describes a member of a pair ofmolecules which have binding specificity for one another. The members ofa specific binding pair may be naturally derived or wholly or partiallysynthetically produced. One member of the pair of molecules has an areaon its surface, or a cavity, which specifically binds to and istherefore complementary to a particular spatial and polar organizationof the other member of the pair of molecules. Thus, the members of thepair have the property of binding specifically to each other. Examplesof types of specific binding pairs include antigen-antibody,Avimerm-substrate, biotin-avidin, hormone-hormone receptor,receptor-ligand, protein-protein, and enzyme-substrate.

The term “adjuvant” refers to a compound or mixture that enhances theimmune response, particularly to an antigen. An adjuvant can serve as atissue depot that slowly releases the antigen and also as a lymphoidsystem activator that non-specifically enhances the immune response(Hood et al., Immunology, Second Ed., 1984, Benjamin/Cummings: MenloPark, Calif., p. 3⁸⁴). Often, a primary challenge with an antigen alone,in the absence of an adjuvant, will fail to elicit a humoral or cellularimmune response. Previously known and utilized adjuvants include, butare not limited to, complete Freund's adjuvant, incomplete Freund'sadjuvant, saponin, mineral gels such as aluminum hydroxide, surfaceactive substances such as lysolecithin, pluronic polyols, polyanions,peptides, oil or hydrocarbon emulsions, keyhole limpet hemocyanins,dinitrophenol, and potentially useful human adjuvant such as BCG(bacille Calmette-Guerin) and Corynebacterium parvum. Mineral saltadjuvants include but are not limited to: aluminum hydroxide, aluminumphosphate, calcium phosphate, zinc hydroxide and calcium hydroxide.Preferably, the adjuvant composition further comprises a lipid of fatemulsion comprising about 10% (by weight) vegetable oil and about 1-2%(by weight) phospholipids. Preferably, the adjuvant composition furtheroptionally comprises an emulsion form having oily particles dispersed ina continuous aqueous phase, having an emulsion forming polyol in anamount of from about 0.2% (by weight) to about 49% (by weight),optionally a metabolizable oil in an emulsion-forming amount of up to15% (by weight), and optionally a glycol ether-based surfactant in anemulsion-stabilizing amount of up to about 5% (by weight).

As used herein, the term “immunomodulator” refers to an agent which isable to modulate an immune response. An example of such modulation is anenhancement of antibody production.

An “immunological response” to a composition or vaccine comprised of anantigen is the development in the host of a cellular- and/orantibody-mediated immune response to the composition or vaccine ofinterest. Usually, such a response consists of the subject producingantibodies, B cells, helper T cells, suppressor T cells, and/orcytotoxic T cells directed specifically to an antigen or antigensincluded in the composition or vaccine of interest.

Molecular Biological Terminology

The term “nucleotide” as used herein refers to a monomeric unit of apolynucleotide that consists of a heterocyclic base, a sugar, and one ormore phosphate groups. The naturally occurring bases (guanine, (G),adenine, (A), cytosine, (C), thymine, (T), and uracil (U)) are typicallyderivatives of purine or pyrimidine, though it should be understood thatnaturally and non-naturally occurring base analogs are also included.The naturally occurring sugar is the pentose (five-carbon sugar)deoxyribose (which forms DNA) or ribose (which forms RNA), though itshould be understood that naturally and non-naturally occurring sugaranalogs are also included. Nucleic acids are typically linked viaphosphate bonds to form nucleic acids, or polynucleotides though manyother linkages are known in the art (such as, though not limited tophosphorothioates, boranophosphates and the like).

The terms “nucleic acid” and “polynucleotide” as used herein refer to apolymeric form of nucleotides of any length, either ribonucleotides(RNA) or deoxyribonucleotides (DNA). These terms refer to the primarystructure of the molecule, and thus include double- and single-strandedDNA, and double- and single-stranded RNA. The terms include, asequivalents, analogs of either RNA or DNA made from nucleotide analogsand modified polynucleotides such as, though not limited to methylatedand/or capped polynucleotides.

A “DNA molecule” refers to the polymeric form of deoxyribonucleotides(adenine, guanine, thymine, or cytosine) in its either single strandedform, or a double-stranded helix. This term refers only to the primaryand secondary structure of the molecule, and does not limit it to anyparticular tertiary forms. Thus, this term includes double-stranded DNAfound, inter alia, in linear DNA molecules (e.g., restrictionfragments), viruses, plasmids, and chromosomes. In discussing thestructure of particular double-stranded DNA molecules, sequences may bedescribed herein according to the normal convention of giving only thesequence in the 5′ to 3′direction along the non transcribed strand ofDNA (i.e., the strand having a sequence homologous to the mRNA).

A DNA “coding sequence” or “coding region” is a double-stranded DNAsequence which is transcribed and translated into a polypeptide in vivowhen placed under the control of appropriate expression controlsequences. The boundaries of the coding sequence (the “open readingframe” or “ORF”) are determined by a start codon at the 5′ (amino)terminus and a translation stop codon at the 3′ (carboxyl) terminus. Acoding sequence can include, but is not limited to, prokaryoticsequences, cDNA from eukaryotic mRNA, genomic DNA sequences fromeukaryotic (e.g., mammalian) DNA, and synthetic DNA sequences. Apolyadenylation signal and transcription termination sequence willusually be located 3′ to the coding sequence. The term “noncodingsequence” or “noncoding region” refers to regions of a polynucleotidesequence that not translated into amino acids (e.g. 5′ and 3′untranslated regions).

The term “reading frame” refers to one of the six possible readingframes, three in each direction, of the double stranded DNA molecule.The reading frame that is used determines which codons are used toencode amino acids within the coding sequence of a DNA molecule.

As used herein, an “antisense” nucleic acid molecule comprises anucleotide sequence which is complementary to a “sense” nucleic acidencoding a protein, e.g., complementary to the coding strand of adouble-stranded cDNA molecule, complementary to an mRNA sequence orcomplementary to the coding strand of a gene. Accordingly, an antisensenucleic acid molecule can hydrogen bond to a sense nucleic acidmolecule.

The term “base pair” or (“bp”): a partnership of adenine (A) withthymine (T), or of cytosine (C) with guanine (G) in a double strandedDNA molecule. In RNA, uracil (U) is substituted for thymine.

As used herein a “codon” refers to the three nucleotides which, whentranscribed and translated, encode a single amino acid residue; or inthe case of WUA, UGA or UAG encode a termination signal. Codons encodingamino acids are well known in the art and are provided for convenienceherein in Table 1.

TABLE 1 Codon Usage Table Codon Amino acid AA Abbrev. Codon Amino acidAA Abbrev. UUU Phenylalanine Phe F UCU Serine Ser S UUC PhenylalaninePhe F UCC Serine Ser S UUA Leucine Leu L UCA Serine Ser S UUG LeucineLeu L UCG Serine Ser S CUU Leucine Leu L CCU Proline Pro P CUC LeucineLeu L CCC Proline Pro P CUA Leucine Leu L CCA Proline Pro P CUG LeucineLeu L CCG Proline Pro P AUU Isoleucine Ile I ACU Threonine Thr T AUCIsoleucine Ile I ACC Threonine Thr T AUA Isoleucine Ile I ACA ThreonineThr T AUG Methionine Met M ACH Threonine Thr T GUU Valine Val V GCUAlanine Ala A GUC Valine Val V GCC Alanine Ala A GUA Valine Val V GCAAlanine Ala A GUG Valine Val V GCG Alanine Ala A UAU Tyrosine Tyr Y UGUCysteine Cys C UAC Tyrosine Tyr Y UGC Cysteine Cys C UUA Stop UGA StopUAG Stop UGG Tryptophan Trp W CAU Histidine His H CGU Arginine Arg R CACHistidine His H CGC Arginine Arg R CAA Glutamine Gln Q CGA Arginine ArgR CAG Glutamine Gln Q CGG Arginine Arg R AAU Asparagine Asn N AGU SerineSer S AAC Asparagine Asn N AGC Serine Ser S AAA Lysine Lys K AGAArginine Arg R AAG Lysine Lys K AGG Arginine Arg R GAU Aspartate Asp DGGU Glycine Gly G GAC Aspartate Asp D GGC Glycine Gly G GAA GlutamateGlu E GGA Glycine Gly G GAG Glutamate Glu E GGG Glycine Gly G

AA: amino acid; Abbr: abbreviation. It should be understood that thecodons specified above are for RNA sequences. The corresponding codonsfor DNA have a T substituted for U. Optimal codon usage is indicated bycodon usage frequencies for expressed genes, for example, as shown inthe codon usage chart from the program “Human—High.cod” from theWisconsin Sequence Analysis Package, Version 8.1, Genetics ComputerGroup, Madison, Wis. Codon usage is also described in, for example, R.Nussinov, “Eukaryotic Dinucleotide Preference Rules and TheirImplications for Degenerate Codon Usage,” J. Mol. Biol. 149: 125-131(1981). The codons which are most frequently used in highly expressedhuman genes are presumptively the optimal codons for expression in humanhost cells and, thus, form the bases for constructing a synthetic codingsequence.

As used herein, a “wobble position” refers to the third position of acodon. Mutations in a DNA molecule within the wobble position of a codontypically result in silent or conservative mutations at the amino acidlevel. For example, there are four codons that encode Glycine, i.e.,GGU, GGC, GGA and GGG, thus mutation of any wobble position nucleotide,to any other nucleotide, does not result in a change at the amino acidlevel of the encoded protein, i.e. is a silent substitution.

Accordingly a “silent substitution” or “silent mutation” is one in whicha nucleotide within a codon is modified, but does not result in a changein the amino acid residue encoded by the codon. Examples includemutations in the third position of a codon, as well as in the firstposition of certain codons, such as the codon “CGG,” which when mutatedto AGG, still encodes the amino acid Arginine (Arg, or R).

The phrase “conservative amino acid substitution” or “conservativemutation” refers to the replacement of one amino acid by another aminoacid with a common property. A functional way to define commonproperties between individual amino acids is to analyze the normalizedfrequencies of amino acid changes between corresponding proteins ofhomologous organisms (Schulz, G. E. and R. H. Schirmer, Principles ofProtein Structure, Springer-Verlag). According to such analyses, groupsof amino acids may be defined where amino acids within a group exchangepreferentially with each other, and therefore resemble each other mostin their impact on the overall protein structure (Schulz, G. E. and R.H. Schirmer, Principles of Protein Structure, Springer-Verlag).

Examples of amino acid groups defined in this manner include: a“charged/polar group,” consisting of Glu, Asp, Asn, Gln, Lys, Arg andHis; an “aromatic, or cyclic group,” consisting of Pro, Phe, Tyr andTrp; and an “aliphatic group” consisting of Gly, Ala, Val, Leu, Ile,Met, Ser, Thr and Cys.

Within each group, subgroups may also be identified, for example, thegroup of charged/polar amino acids may be sub-divided into thesub-groups consisting of the “positively-charged sub-group,” consistingof Lys, Arg and His; the negatively-charged sub-group,” consisting ofGlu and Asp, and the “polar sub-group” consisting of Asn and Gln.

The aromatic, or cyclic group may be sub-divided into the sub-groupsconsisting of the “nitrogen ring sub-group,” consisting of Pro, His andTrp; and the “phenyl sub-group” consisting of Phe and Tyr.

The aliphatic group may be sub-divided into the sub-groups consisting ofthe “large aliphatic non-polar sub-group,” consisting of Val, Leu andIle; the “aliphatic slightly-polar sub-group,” consisting of Met, Ser,Thr and Cys; and the “small-residue sub-group,” consisting of, Gly, andAla.

Examples of conservative mutations include amino acid substitutions ofamino acids within the sub-groups above, for example, Lys for Arg andvice versa such that a positive charge may be maintained; Glu for Aspand vice versa such that a negative charge may be maintained; Ser forThr such that a free —OH can be maintained; and Gln for Asn such that afree —NH2 can be maintained.

“Semi-conservative mutations” include amino acid substitutions of aminoacids with the same groups listed above, that do not share the samesub-group. For example, the mutation of Asp for Asn, or Asn for Lys allinvolve amino acids within the same group, but different sub-groups.

“Non-conservative mutations” involve amino acid substitutions betweendifferent groups, for example Lys for Leu, or Phe for Ser, etc.

The term “amino acid residue” refers to the radical derived from thecorresponding alpha-amino acid by eliminating the OH portion of thecarboxyl group and the H-portion of the alpha amino group. For the mostpart, the amino acids used in the application are those naturallyoccurring amino acids found in proteins, or the naturally occurringanabolic or catabolic products of such amino acids which contain aminoand carboxyl groups. Alternatively, un-natural amino acids can beincorporated into proteins to facilitate the chemical conjugation toother proteins, toxins, small organic compounds or anti-cancer agents(Datta et al., J Am Chem. Soc. (2002) 124 (20):5652-3). In general, theabbreviations used herein for designating the amino acids and theprotective groups are based on recommendations of the IUPAC-IUBCommission on Biochemical Nomenclature (see Biochemistry (1972) 11:1726-1732). The term “amino acid residue” also includes analogs,derivatives and congeners of any specific amino acid referred to herein,as well as C-terminal or N-terminal protected amino acid derivatives(e.g., modified with an N-terminal or C-terminal protecting group). Forexample, the present invention contemplates the use of amino acidanalogs wherein a side chain is lengthened or shorted while stillproviding a carboxyl, amino or other reactive precursor functional groupfor cyclization, as well as amino acid analogs having variant sidechains with appropriate functional groups).

The term “amino acid side chain” is that part of an amino acid exclusiveof the —CH—(NH₂)COOH portion, as defined by K. D. Kopple, “Peptides andAmino Acids,” W. A. Benjamin Inc., New York and Amsterdam, 1996, pages 2and 33; examples of such side chains of the common amino acids are—CH₂CH₂SCH₃ (the side chain of methionine), —CH₂(CH₃)—CH₂CH₃ (the sidechain of isoleucine), —CH₂CH(CH₃)₂ (the side chain of leucine) or H—(theside chain of glycine).

The amino acid residues described herein are preferred to be in the “L”isomeric form. However, residues in the “D” isomeric form can besubstituted for any L-amino acid residue, as long as the desiredfunctional property of antibody (immunoglobulin)-binding is retained bythe polypeptide. NH₂ refers to the free amino group present at the aminoterminus of a polypeptide. COOH refers to the free carboxy group presentat the carboxy terminus of a polypeptide.

An “amino acid motif” is a sequence of amino acids, optionally a genericset of conserved amino acids, associated with a particular functionalactivity.

As used herein, the terms “protein,” “peptide” and “polypeptide” areused interchangeably to refer to polymers of amino acid residues of anylength connected to one another by peptide bonds between the alpha-aminogroup and carboxy group of contiguous amino acid residues. Polypeptides,proteins and peptides may exist as linear polymers, branched polymers orin circular form. These terms also include forms that arepost-translationally modified in vivo, or chemically modified duringsynthesis.

It should be noted that all amino-acid residue sequences are representedherein by formulae whose left and right orientation is in theconventional direction of amino-terminus to carboxy terminus.Furthermore, it should be noted that a dash at the beginning or end ofan amino acid residue sequence indicates a peptide bond to a furthersequence of one or more amino-acid-residues.

The terms “gene,” “recombinant gene” and “gene construct” as usedherein, refer to a DNA molecule, or portion of a DNA molecule, thatencodes a protein. The DNA molecule can contain an open reading frameencoding the protein (as exon sequences) and can further include intronsequences. The term “intron” as used herein, refers to a DNA sequencepresent in a given gene which is not translated into protein and isgenerally found between exons. Usually, it is desirable for the gene tobe operably linked to, (or it may comprise), one or more promoters,enhancers, repressors and/or other regulatory sequences to modulate theactivity or expression of the gene, as is well known in the art.

As used herein, a “complementary DNA” or “cDNA” includes recombinantpolynucleotides synthesized by reverse transcription of mRNA and fromwhich intervening sequences (introns) have been removed.

The term “operably linked” as used herein, describes the relationshipbetween two polynucleotide regions such that they are functionallyrelated or coupled to each other. For example, a promoter (or otherexpression control sequence) is operably linked to a coding sequence ifit controls (and is capable of effecting) the transcription of thecoding sequence. Although an operably linked promoter is generallylocated upstream of the coding sequence, it is not necessarilycontiguous with it.

“Expression control sequences” are DNA regulatory sequences, such aspromoters, enhancers, polyadenylation signals, terminators, internalribosome entry sites (IRES) and the like, that provide for theexpression of a coding sequence in a host cell. Exemplary expressioncontrol sequences are described in Goeddel; Gene Expression Technology:Methods in Enzymology 185, Academic Press, San Diego, Calif. (1990).

A “promoter” is a DNA regulatory region capable of binding RNApolymerase in a cell and initiating transcription of a downstream (3′direction) coding sequence. As used herein, the promoter sequence isbounded at its 3′ terminus by the transcription initiation site andextends upstream (5′ direction) to include the minimum number of basesor elements necessary to initiate transcription at levels detectableabove background. Within the promoter sequence will be found atranscription initiation site (conveniently defined by mapping withnuclease 5′), as well as protein binding domains (consensus sequences)responsible for the binding of RNA polymerase. Eukaryotic promoters willoften, but not always, contain “TATA” boxes and “CAT” boxes. Prokaryoticpromoters contain Shine-Dalgamo sequences in addition to the −10 and −35consensus sequences.

A large number of promoters, including constitutive, inducible andrepressible promoters, from a variety of different sources are wellknown in the art. Representative sources include for example, viral,mammalian, insect, plant, yeast, and bacterial cell types), and suitablepromoters from these sources are readily available, or can be madesynthetically, based on sequences publicly available on line or, forexample, from depositories such as the ATCC as well as other commercialor individual sources. Promoters can be unidirectional (i.e., initiatetranscription in one direction) or bi-directional (i.e., initiatetranscription in either a 3′ or 5′ direction). Non-limiting examples ofpromoters include, for example, the T7 bacterial expression system, pBAD(araa) bacterial expression system, the cytomegalovirus (CMV) promoter,the SV40 promoter, the RSV promoter. Inducible promoters include the Tetsystem, (U.S. Pat. Nos. 5,464,758 and 5,814,618), the Ecdysone induciblesystem (No et al., Proc. Natl. Acad. Sci. (1996) 93 (8) 3346-3351; theT-RE_(x)™ system (Invitrogen Carlsbad, Calif.), LacSwitch® (Stratagene,(San Diego, Calif.) and the Cre-ERT tamoxifen inducible recombinasesystem (Indra et al. Nuc. Acid. Res. (1999) 27 (22)-432-44327; Nuc.Acid. Res. (2000) 28 (23) e99; U.S. Pat. No. 7,112,715). See generally,Kramer & Fussenegger Methods Mol. Biol. (2005) 308 123-144) or anypromoter known in the art suitable for expression in the desired cells.

As used herein, a “minimal promoter” refers to a partial promotersequence which defines the transcription start site but which by itselfis not capable, if at all, of initiating transcription efficiently. Theactivity of such minimal promoters depends on the binding of activatorssuch as a tetracycline-controlled transactivator to operably linkedbinding sites.

The terms “IRES” or “internal ribosome entry site” refer to apolynucleotide element that acts to enhance the translation of a codingsequence encoded with a. polycistronic messenger RNA. IRES elements,mediate the initiation of translation by directly recruiting and bindingribosomes to a messenger RNA (mRNA) molecule, bypassing the 7-methylguanosine-cap involved in typical ribosome scanning. The presence of anIRES sequence can increase the level of cap-independent translation of adesired protein. Early publications descriptively refer to IRESsequences as “translation enhancers.” For example, cardioviral RNA“translation enhancers” are described in U.S. Pat. No. 4,937,190 toPalmenberg et al. and U.S. Pat. No. 5,770,428 to Boris-Lawrie.

The terms “nuclear localization signal” and “NLS” refer to a domain, ordomains capable of mediating the nuclear import of a protein orpolynucleotide, or retention thereof, within the nucleus of a cell. A“strong nuclear import signal” represents a domain or domains capable ofmediating greater than 90% subcellular localization in the nucleus whenoperatively linked to a protein of interest. Representative examples ofNLSs include but are not limited to, monopartite nuclear localizationsignals, bipartite nuclear localization signals and N and C-terminalmotifs. N terminal basic domains usually conform to the consensussequence K-K/R-X-KJR which was first discovered in the SV40 large Tantigen and which represents a monopartite NLS. One non-limiting exampleof an N-terminal basic domain NLS is PKKKRKV (SEQ ID NO: 439). Alsoknown are bipartite nuclear localization signals which contain twoclusters of basic amino acids separated by a spacer of about 10 aminoacids, as exemplified by the NLS from nucleoplasmin: KR[PAATKKAGQA]KKKK(SEQ ID NO: 450). N and C-terminal motifs include, for example, theacidic M9 domain of hnRNP A1, the sequence KIPIK (SEQ ID NO: 464) inyeast transcription repressor Matα2 and the complex signals of U snRNPs.Most of these NLSs appear to be recognized directly by specificreceptors of the importin β family.

The term “enhancer” as used herein, refers to a DNA sequence thatincreases transcription of, for example, a gene or coding sequence towhich it is operably linked. Enhancers can be located many kilobasesaway from the coding sequence and can mediate the binding of regulatoryfactors, patterns of DNA methylation or changes in DNA structure. Alarge number of enhancers, from a variety of different sources are wellknown in the art and available as or within cloned polynucleotides(from, e.g., depositories such as the ATCC as well as other commercialor individual sources). A number of polynucleotides comprising promoters(such as the commonly-used CMV promoter) also comprise enhancersequences. Operably linked enhancers can be located upstream, within, ordownstream of coding sequences. The term “Ig enhancers” refers toenhancer elements derived from enhancer regions mapped within the Iglocus (such enhancers include for example, the heavy chain (mu) 5′enhancers, light chain (kappa) 5′ enhancers, kappa and mu intronicenhancers, and 3′ enhancers, (see generally Paul WE (ed) FundamentalImmunology, 3^(rd) Edition, Raven Press, New York (1993) pages 353-363;U.S. Pat. No. 5,885,827).

“Terminator sequences” are those that result in termination oftranscription. Termination sequences are known in the art and include,but are not limited to, poly A (e.g., Bgh Poly A and SV40 Poly A)terminators. A transcriptional termination signal will typically includea region of 3′untranslated region (or “3′ ut”), an optional intron (alsoreferred to as intervening sequence or “IVS”) and one or more polyadenylation signals (“p(A)” or “pA.” Terminator sequences may also bereferred to as “IVS-pA,” “IVS+p(A),” “3′ ut+p(A)” or “3′ ut/p(A).”Natural or synthetic terminators can be used as a terminator region.

The terms “polyadenylation,” “polyadenylation sequence” and“polyadenylation signal”, “Poly A,” “p(A)” or “pA” refer to a nucleicacid sequence present in a RNA transcript that allows for thetranscript, when in the presence of the polyadenyl transferase enzyme,to be polyadenylated. Many polyadenylation signals are known in the art.Non-limiting examples include the human variant growth hormonepolyadenylation signal, the SV40 late polyadenylation signal and thebovine growth hormone polyadenylation signal.

The term “splice site” as used herein refers to polynucleotides that arecapable of being recognized by the spicing machinery of a eukaryoticcell as suitable for being cut and/or ligated to a corresponding splicesite. Splice sites allow for the excision of introns present in apre-mRNA transcript. Typically the 5′ portion of the splice site isreferred to as the splice donor and the 3′ corresponding splice site isreferred to as the acceptor splice site. The term splice site includes,for example, naturally occurring splice sites, engineered splice sites,for example, synthetic splice sites, canonical or consensus splicesites, and/or non-canonical splice sites, for example, cryptic splicesites.

A “signal sequence” can be included before the coding sequence. Thissequence encodes a signal peptide, N-terminal to the polypeptide, thatcommunicates to the host cell to direct the polypeptide to the cellsurface or secrete the polypeptide into the media, and this signalpeptide is clipped off by the host cell before the protein leaves thecell. Signal sequences can be found associated with a variety ofproteins native to prokaryotes and eukaryotes.

“Post-translational modification” can encompass any one of or acombination of modifications including covalent modification, which aprotein undergoes after translation is complete and after being releasedfrom the ribosome or on the nascent polypeptide co-translationally.Posttranslational modification includes but is not limited tophosphorylation, myristylation, ubiquitination, glycosylation, coenzymeattachment, methylation, S-nitrosylation and acetylation.Posttranslational modification can modulate or influence the activity ofa protein, its intracellular or extracellular destination, its stabilityor half-life, and/or its recognition by ligands, receptors or otherproteins. Post-translational modification can occur in cell organelles,in the nucleus or cytoplasm or extracellularly.

The term “primer” as used herein refers to an oligonucleotide, whetheroccurring naturally as in a purified restriction digest or producedsynthetically, which is capable of acting as a point of initiation ofsynthesis when placed under conditions in which synthesis of a primerextension product, which is complementary to a nucleic acid strand, isinduced, i.e., in the presence of nucleotides and an inducing agent suchas a DNA polymerase and at a suitable temperature and pH. The primer maybe either single-stranded or double-stranded and must be sufficientlylong to prime the synthesis of the desired extension product in thepresence of the inducing agent. The exact length of the primer willdepend upon many factors, including temperature, source of primer anduse of the method. For example, for diagnostic applications, dependingon the complexity of the target sequence, the oligonucleotide primertypically contains 15-25 or more nucleotides, although it may containfewer nucleotides. The polynucleotide primers can be prepared using anysuitable method, such as, for example, the phosphotriester onphosphodiester methods see Narang et al., Meth. Enzymol., 68:90, (1979);U.S. Pat. No. 4,356,270; and Brown et al., Meth. Enzymol., 68:109,(1979).

The primers herein are selected to be “substantially” complementary todifferent strands of a particular target polynucleotide sequence. Thismeans that the primers must be sufficiently complementary to hybridizewith their respective strands. Therefore, the primer sequence need notreflect the exact sequence of the template. For example, anon-complementary nucleotide fragment may be attached to the 5′ end ofthe primer, with the remainder of the primer sequence beingcomplementary to the strand. Alternatively, non-complementary bases orlonger sequences can be interspersed into the primer, provided that theprimer sequence has sufficient complementarity with the sequence of thestrand to hybridize therewith and thereby form the template for thesynthesis of the extension product.

As used herein, the terms “restriction endonucleases” and “restrictionenzymes” refer to bacterial enzymes, each of which cut double-strandedDNA at or near a specific nucleotide sequence.

The term “multiple cloning site” as used herein, refers to a segment ofa vector polynucleotide which can recognize one or more differentrestriction enzymes.

A “replicon” is any genetic element (e.g., plasmid, episome, chromosome,yeast artificial chromosome (YAC), or virus) that functions as anautonomous unit of DNA replication in vivo; i.e., capable of replicationunder its own control, and containing autonomous replicating sequences.

A “vector” or “cloning vector” is a replicon, such as plasmid, phage orcosmid, into which another polynucleotide segment may be introduced soas to bring about the replication of the inserted segment. Vectorstypically exist as circular, double stranded DNA, and range in size forma few kilobases (kb) to hundreds of kb. Preferred cloning vectors havebeen modified from naturally occurring plasmids to facilitate thecloning and recombinant manipulation of polynucleotide sequences. Manysuch vectors are well known in the art; see for example, by Sambrook(In. “Molecular Cloning: A Laboratory Manual,” second edition, edited bySambrook, Fritsch, & Maniatis, Cold Spring Harbor Laboratory, (1989)),Maniatis, In: Cell Biology: A Comprehensive Treatise, Vol. 3, GeneSequence Expression, Academic Press, NY, pp. 563-608 (1980).

The term “expression vector” as used herein, refers to an agent used forexpressing certain polynucleotides within a host cell or in-vitroexpression system. The term includes plasmids, episomes, cosmidsretroviruses or phages; the expression vector can be used to express aDNA sequence encoding a desired protein and in one aspect includes atranscriptional unit comprising an assembly of expression controlsequences. The choice of promoter and other regulatory elementsgenerally varies according to the intended host cell, or in-vitroexpression system.

An “episomal expression vector” is able to replicate in the host cell,and persists as an extrachromosomal segment of DNA within the host cellin the presence of appropriate selective pressure. (See for example,Conese et al., Gene Therapy 11 1735-1742 (2004)). Representativecommercially available episomal expression vectors include, but are notlimited to, episomal plasmids that utilize Epstein Barr Nuclear Antigen1 (EBNA1) and the Epstein Barr Virus (EBV) origin of replication (oriP).The vectors pREP4, pCEP4, pREP7 from Invitrogen, pcDNA3.1 fromInvitrogen, and pBK-CMV from Stratagene represent non-limiting examplesof an episomal vector that uses T-antigen and the SV40 origin ofreplication in lieu of EBNA1 and oriP.

An “integrating expression vector” may randomly integrate into the hostcell's DNA, or may include a recombination site to enable the specificrecombination between the expression vector and the host cellschromosome. Such integrating expression vectors may utilize theendogenous expression control sequences of the host cell's chromosomesto effect expression of the desired protein. Examples of vectors thatintegrate in a site specific manner include, for example, components ofthe flp-in system from Invitrogen (e.g., pcDNAm5/FRT), or the cre-loxsystem, such as can be found in the pExchange-6 Core Vectors fromStratagene. Examples of vectors that integrate into host cellchromosomes in a random fashion include, for example, pcDNA3.1 (whenintroduced in the absence of T-antigen) from Invitrogen, pCI or pFN10A(ACT) Flexi® from Promega.

Representative commercially available viral expression vectors include,but are not limited to, the adenovirus-based Per.C6 system availablefrom Crucell, Inc., the lentiviral-based pLPI from Invitrogen, and theRetroviral Vectors pFB-ERV plus pCFB-EGSH from Stratagene.

Alternatively, the expression vector may be used to introduce andintegrate a strong promoter or enhancer sequences into a locus in thecell so as to modulate the expression of an endogenous gene of interest(Capecchi M R. Nat Rev Genet. (2005); 6 (6):507-12; Schindehutte et al.,Stem Cells (2005); 23 (1):10⁻⁵). This approach can also be used toinsert an inducible promoter, such as the Tet-On promoter (U.S. Pat.Nos. 5,464,758 and 5,814,618), in to the genomic DNA of the cell so asto provide inducible expression of an endogenous gene of interest. Theactivating construct can also include targeting sequence(s) to enablehomologous or non-homologous recombination of the activating sequenceinto a desired locus specific for the gene of interest (see for example,Garcia-Otin & Guillou, Front Biosci. (2006) 11:1108-36). Alternatively,an inducible recombinase system, such as the Cre-ER system, can be usedto activate a transgene in the presence of 4-hydroxytamoxifen. (Indra etal. Nuc. Acid. Res. (1999) 27 (22) 4324-4327; Nuc. Acid. Res. (2000) 28(23) e99; U.S. Pat. No. 7,112,715). Expression vectors may also includeanti-sense, ribozymes or siRNA polynucleotides to reduce the expressionof target sequences. (See generally, Sioud M, & Iversen, Curr. DrugTargets (2005) 6 (6):647-53; Sandy et al., Biotechniques (2005) 39(2):215-24).

As used herein, a “recombination system” refers to one which allows forrecombination between a vector of the present application and achromosome for incorporation of a gene of interest. Recombinationsystems are known in the art and include, for example, Cre/Lox systemsand FLP-IN systems.

As used herein an “in-vitro expression system” refers to cell freesystems that enable the transcription, or coupled transcription andtranslation of DNA templates. Such systems include for example theclassical rabbit reticulocyte system, as well as novel cell freesynthesis systems, (J. Biotechnol. (2004) 110 (3) 257-63; BiotechnolAnnu. Rev. (2004) 101-30).

As used herein, a “Cre/Lox” system refers to one such as described byAbremski et al., Cell, 32: 1301-1311 (1983) for a site-specificrecombination system of bacteriophage P1. Methods of using Cre-Loxsystems are known in the art; see, for example, U.S. Pat. No. 4,959,317,which is hereby incorporated in its entirety by reference. The systemconsists of a recombination site designated loxP and a recombinasedesignated Cre. In methods for producing site-specific recombination ofDNA in eukaryotic cells, DNA sequences having first and second lox sitesare typically introduced into eukaryotic cells and contacted with Cre,thereby producing recombination at the lox sites.

As used here, “FLP-IN” recombination refers to systems in which apolynucleotide activation/inactivation and site-specific integrationsystem has been developed for mammalian cells. The system is based onthe recombination of transfected sequences by FLP, a recombinase derivedfrom Saccharomyces. In several cell lines, FLP has been shown to rapidlyand precisely recombine copies of its specific target sequence. FLP-INsystems have been described in, for example, U.S. Pat. Nos. 5,654,182and 5,677,177).

The term “transfection,” “transformation,” or “transduction” as usedherein, refers to the introduction of one or more exogenouspolynucleotides into a host cell by using one or physical or chemicalmethods. Many transfection techniques are known to those of ordinaryskill in the art including but not limited to calcium phosphate DNAco-precipitation (see Methods in Molecular Biology, Vol. 7, GeneTransfer and Expression Protocols, Ed. E. J. Murray, Humana Press(1991)); DEAE-dextran; electroporation; cationic liposome-mediatedtransfection; tungsten particle-facilitated microparticle bombardment(Johnston, S. A., Nature 346: 776-777 (1990)); and strontium phosphateDNA co-precipitation (Brash D. E. et al. Molec. Cell. Biol. 7: 2031-2034(1987). Phage or retroviral vectors can be introduced into host cells,after growth of infectious particles in packaging cells that arecommercially available.

The terms “cells,” “cell cultures,” “cell line,” “recombinant hostcells,” “recipient cells” and “host cells” are often usedinterchangeably and will be clear from the context in which they areused. These terms include the primary subject cells and any progenythereof, without regard to the number of transfers. It should beunderstood that not all progeny are exactly identical to the parentalcell (due to deliberate or inadvertent mutations or differences inenvironment). However, such altered progeny are included in these terms,so long as the progeny retain the same functionality as that of theoriginally transformed cell. For example, though not limited to, such acharacteristic might be the ability to produce a particular recombinantprotein. A “mutator positive cell line” is a cell line containingcellular factors that are sufficient to work in combination with othervector elements to effect hypermutation. The cell line can be any ofthose known in the art or described herein. A “clone” is a population ofcells derived from a single cell or common ancestor by mitosis.

A “reporter gene” refers to a polynucleotide that confers the ability tobe specifically detected, (or detected and selected) typically whenexpressed with a cell of interest. Numerous reporter gene systems areknown in the art and include, for example alkaline phosphatase (Berger,J., et al., Gene 661-10 (1988); Kain, S R., Methods Mol. Biol. 63 49-60(1997)), beta-galactosidase (U.S. Pat. No. 5,070,012), chloramphenicolacetyltransferase (Gorman et al., Mol. Cell. Biol. 2 1044-51 (1982)),beta glucuronidase, peroxidase, beta lactamase (U.S. Pat. Nos.5,741,657, 5,955,604), catalytic antibodies, luciferases (U.S. Pat. Nos.5,221,623; 5,683,888; 5,674,713; 5,650,289; 5,843,746) and naturallyfluorescent proteins (Tsien, R Y, Annu. Rev. Biochem. 67 509-544(1998)). The term “reporter gene,” also includes any peptide which canbe specifically detected based on the use of one or more, antibodies,epitopes, binding partners, substrates, modifying enzymes, receptors, orligands that are capable of, or desired to (or desired not to), interactwith the peptide of interest to create a detectable signal. Reportergenes also include genes that can modulate cellular phenotype.

The term “selectable marker gene” as used herein, refers topolynucleotides that allow cells carrying the polynucleotide to bespecifically selected for or against, in the presence of a correspondingselective agent. Selectable markers can be positive, negative orbifunctional. Positive selectable markers allow selection for cellscarrying the marker, whereas negative selectable markers allow cellscarrying the marker to be selectively eliminated. The selectable markerpolynucleotide can either be directly linked to the polynucleotides tobe expressed, or introduced into the same cell by co-transfection. Avariety of such marker polynucleotides have been described, includingbifunctional (i.e., positive/negative) markers (see, e.g., WO 92/08796,published May 29, 1992, and WO 94/28143, published Dec. 8, 1994), herebyincorporated in their entirety by reference herein. Specific examples ofselectable markers of drug-resistance genes include, but are not limitedto, ampicillin, tetracycline, blasticidin, puromycin, hygromycin,ouabain or kanamycin. Specific examples of selectable markers are those,for example, that encode proteins that confer resistance to cytostaticor cytocidal drugs, such as the DHFR protein, which confers resistanceto methotrexate (Wigler et al., Proc. Natl. Acad. Sci. USA, 77:3567(1980); O'Hare et al., Proc. Natl. Acad. Sci. USA, 78:1527 (1981)); theGPF protein, which confers resistance to mycophenolic acid (Mulligan &Berg, Proc. Natl. Acad. Sci. USA, 78:2072 (1981)), the neomycinresistance marker, which confers resistance to the aminoglycoside G-418(Colberre-Garapin et al., J. Mol. Biol., 150:1 (1981)); the Hygromycinprotein, which confers resistance to hygromycin (Santerre et al., Gene,30:147 (1984)); murine Na+, K+-ATPase alpha subunit, which confersresistance to ouabain (Kent et al., Science, 237:901-903 (1987); and theZeocin™ resistance marker (available commercially from Invitrogen). Inaddition, the herpes simplex virus thymidine kinase (Wigler et al.,Cell, 11:223 (1977)), hypoxanthine-guanine phosphoribosyltransferase(Szybalska & Szybalski, Proc. Natl. Acad. Sci. USA, 48:2026 (1962)), andadenine phosphoribosyltransferase (Lowy et al., Cell, 22:817 (1980)) canbe employed in tk−, hgprt− or aprt− cells, respectively. Glutaminesynthetase permits the growth of cells in glutamine(GS)-free media (see,e.g., U.S. Pat. Nos. 5,122,464; 5,770,359; and 5,827,739). Otherselectable markers encode, for example, puromycin N-acetyl transferaseor adenosine deaminase.

“Homology” or “identity” or “similarity” refers to sequence similaritybetween two peptides or between two nucleic acid molecules. Homology andidentity can each be determined by comparing a position in each sequencewhich may be aligned for purposes of comparison. When an equivalentposition in the compared sequences is occupied by the same base or aminoacid, then the molecules are identical at that position; when theequivalent site occupied by the same or a similar amino acid residue(e.g., similar in steric and/or electronic nature), then the moleculescan be referred to as homologous (similar) at that position. Expressionas a percentage of homology/similarity or identity refers to a functionof the number of identical or similar amino acids at positions shared bythe compared sequences. A sequence which is “unrelated” or“non-homologous” shares less than 40% identity, less than 35% identity,less than 30% identity, or less than 25% identity with a sequence of thepresent invention. In comparing two sequences, the absence of residues(amino acids or nucleic acids) or presence of extra residues alsodecreases the identity and homology/similarity.

The term “homology” describes a mathematically based comparison ofsequence similarities which is used to identify genes or proteins withsimilar functions or motifs. The nucleic acid and protein sequences ofthe present invention may be used as a “query sequence” to perform asearch against public databases to, for example, identify other familymembers, related sequences or homologs. Such searches can be performedusing the NBLAST and XBLAST programs (version 2.0) of Altschul, et al.(1990) J. Mol. Biol. 215:403-10. BLAST nucleotide searches can beperformed with the NBLAST program, score=100, wordlength=12 to obtainnucleotide sequences homologous to nucleic acid molecules of theinvention. BLAST protein searches can be performed with the XBLASTprogram, score=50, wordlength=3 to obtain amino acid sequenceshomologous to protein molecules of the invention. To obtain gappedalignments for comparison purposes, Gapped BLAST can be utilized asdescribed in Altschul et al., (1997) Nucleic Acids Res.25(17):3389-3402. When utilizing BLAST and Gapped BLAST programs, thedefault parameters of the respective programs (e.g., XBLAST and BLAST)can be used (See www.ncbi.nlm.nih.gov).

As used herein, “identity” means the percentage of identical nucleotideor amino acid residues at corresponding positions in two or moresequences when the sequences are aligned to maximize sequence matching,i.e., taking into account gaps and insertions. Identity can be readilycalculated by known methods, including but not limited to thosedescribed in (Computational Molecular Biology, Lesk, A. M., ed., OxfordUniversity Press, New York, 1988; Biocomputing: Informatics and GenomeProjects, Smith, D. W., ed., Academic Press, New York, 1993; ComputerAnalysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G.,eds., Humana Press, New Jersey, 1994; Sequence Analysis in MolecularBiology, von Heinje, G., Academic Press, 1987; and Sequence AnalysisPrimer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York,1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073(1988). Methods to determine identity are designed to give the largestmatch between the sequences tested. Moreover, methods to determineidentity are codified in publicly available computer programs. Computerprogram methods to determine identity between two sequences include, butare not limited to, the GCG program package (Devereux, J., et al.,Nucleic Acids Research 12(1): 387 (1984)), BLASTP, BLASTN, and FASTA(Altschul, S. F. et al., J. Molec. Biol. 215: 403-410 (1990) andAltschul et al. Nuc. Acids Res. 25: 3389-3402 (1997)). The BLAST Xprogram is publicly available from NCBI and other sources (BLAST Manual,Altschul, S., et al., NCBI NLM NIH Bethesda, Md. 20894; Altschul, S., etal., J. Mol. Biol. 215: 403-410 (1990). The well known Smith Watermanalgorithm may also be used to determine identity.

A “heterologous” region of the DNA construct is an identifiable segmentof DNA within a larger DNA molecule that is not found in associationwith the larger molecule in nature. Thus, when the heterologous regionencodes a mammalian gene, the gene will usually be flanked by DNA thatdoes not flank the mammalian genomic DNA in the genome of the sourceorganism. Another example of a heterologous coding sequence is aconstruct where the coding sequence itself is not found in nature (e.g.,a cDNA where the genomic coding sequence contains introns, or syntheticsequences having codons different than the native gene). Allelicvariations or naturally-occurring mutational events do not give rise toa heterologous region of DNA as defined herein.

SHM Related Terminology

The term “activation-induced cytidine deaminase” or (“AID”) refers tomembers of the AID/APOBEC family of RNA/DNA editing cytidine deaminasescapable of mediating the deamination of cytosine to uracil within a DNAsequence. (See generally Conticello et al., Mol. Biol. Evol. 22 No 2367-377 (2005), Evolution of the AID/APOBEC Family of Polynucleotide(Deoxy)cytidine Deaminases); U.S. Pat. No. 6,815,194). Suitable AIDenzymes include all vertebrate forms of the enzyme, including, forexample, primate, rodent, avian and bony fish. Representative examplesof AID enzymes include without limitation, human (accession No.NP_(—)065712), rat, chicken, canine and mouse (accession No.NP_(—)033775) forms. In one embodiment, AID enzymes include the mutationL198A.

The term “AID homolog” refers to the enzymes of the Apobec family andinclude, for example, Apobec-1, Apobec3C or Apobec3G (described, forexample, by Jarmuz et al., (2002) Genomics, 79: 285-296) (2002)). AIDand AID homologs further include, without limitation, modifiedpolypeptides, or portions thereof, which retain the activity of a nativeAID/APOBEC polypeptides (e.g. mutants or muteins) that retain theability to deaminate a polynucleotide sequence. The term “AID activity”includes activity mediated by AID and AID homologs.

The term “substrate for SHM” refers to a synthetic or semi-syntheticpolynucleotide sequence which is acted upon by AID and/or error proneDNA polymerases to effect a change in the nucleic acid sequence of thesynthetic or semi-synthetic polynucleotide sequence.

The term “transition mutations” refers to base changes in a DNA sequencein which a pyrimidine (cytidine (C) or thymidine (T) is replaced byanother pyrimidine, or a purine (adenosine (A) or guanosine (G) isreplaced by another purine.

The term “transversion mutations” refers to base changes in a DNAsequence in which a pyrimidine (cytidine (C) or thymidine (T) isreplaced by a purine (adenosine (A) or guanosine (G), or a purine isreplaced by a pyrimidine.

The term “base excision repair” refers to a DNA repair pathway thatremoves single bases from DNA such as uridine nucleotides arising bydeamination of cytidine. Repair is initiated by uracil glycosylase thatrecognizes and removes uracil from single- or double-stranded DNA toleave an abasic site.

The term “mismatch repair” refers to the repair pathway that recognizesand corrects mismatched bases, such as those that typically arise fromerrors of chromosomal DNA replication.

As used herein, the term “SHM hot spot” or “hot spot” refers to apolynucleotide sequence, or motif, of 3-6 nucleotides that exhibits anincreased tendency to undergo somatic hypermutation, as determined via astatistical analysis of SHM mutations in antibody genes (see Tables 2and 3 which provide a relative ranking of various motifs for SHM, andTable 6 which lists canonical hot spots and cold spots). The statisticalanalysis can be extrapolated to analysis of SHM mutations innon-antibody genes as described elsewhere herein. For the purposes ofgraphical representations of hot spots in Figures, the first nucleotideof a canonical hot spot is represented by the letter “H.”

Likewise, as used herein, a “SHM coldspot” or “cold spot” refers to apolynucleotide or motif, of 3-6 nucleotides that exhibits a decreasedtendency to undergo somatic hypermutation, as determined via astatistical analysis of SHM mutations in antibody genes (see Tables 2and 3 which provide a relative ranking of various motifs for SHM, andTable 6 which lists canonical hot spots and cold spots). The statisticalanalysis can be extrapolated to analysis of SHM mutations innon-antibody genes as described elsewhere herein. For the purposes ofgraphical representations of cold spots in Figures, the first nucleotideof a canonical cold spot is represented by the letter “C.”

The term “somatic hypermutation motif” or “SHM motif” refers to apolynucleotide sequence that includes, or can be altered to include, oneor more hot spots or cold spots, and which encodes a defined set ofamino acids. SHM motifs can be of any size, but are conveniently basedaround polynucleotides of about 2 to about 20 nucleotides in size, orfrom about 3 to about 9 nucleotides in size. SHM motifs can include anycombination of hot spots and cold spots, or may lack both hot spots andcold spots.

The term “preferred SHM motif” refers to an SHM motif that includes oneor more preferred (canonical) SHM codons (See Table 6 and Table 9infra).

The terms “preferred hot spot SHM codon,” “preferred hot spot SHM motif”“preferred SHM hot spot codon” and “preferred SHM hot spot motif,” allrefer to a codon including, but not limited to codons AAC, TAC, TAT,AGT, or AGC. Such sequences may be potentially embedded within thecontext of a larger SHM motif, recruits SHM mediated mutagenesis andgenerates targeted amino acid diversity at that codon.

As used herein, a polynucleotide sequence has been “optimized for SHM”if the polynucleotide, or a portion thereof has been altered to increaseor decrease the frequency and/or location of hot spots and/or cold spotswithin the polynucleotide. A polynucleotide that has been made“susceptible to SHM” if the polynucleotide, or a portion thereof, hasbeen altered to increase the frequency and/or location of hot spotswithin the polynucleotide or to decrease the frequency (density) and/orlocation of cold spots within the polynucleotide. Conversely, apolynucleotide sequence has been made “resistant to SHM” if thepolynucleotide sequence, or a portion thereof, has been altered todecrease the frequency (density) and/or location of hot spots within theopen reading frame of the polynucleotide sequence. In general, asequence can be prepared that has a greater or lesser propensity toundergo SHM mediated mutagenesis by altering the codon usage, and/or theamino acids encoded by polynucleotide sequence.

Optimization of a polynucleotide sequence refers to modifying about 1%,about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 25%,about 50%, about 75%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, 100% or any range therein of the nucleotides in thepolynucleotide sequence. Optimization of a polynucleotide sequence alsorefers to modifying about 1, about 2, about 3, about 4, about 5, about10, about 20, about 25, about 50, about 75, about 90, about 95, about96, about 97, about 98, about 99, about 100, about 200, about 300, about400, about 500, about 750, about 1000, about 1500, about 2000, about2500, about 3000 or more, or any range therein of the nucleotides in thepolynucleotide sequence such that some or all of the nucleotides areoptimized for SHM-mediated mutagenesis. Reduction in the frequency(density) of hot spots and/or cold spots refers to reducing about 1%,about 2%, about 3%, about 4%, about 5%, about 10%, about 20%, about 25%,about 50%, about 75%, about 90%, about 95%, about 96%, about 97%, about98%, about 99%, 100% or any range therein of the hot spots or cold spotsin a polynucleotide sequence. Increasing the frequency (density) of hotspots and/or cold spots refers to increasing about 1%, about 2%, about3%, about 4%, about 5%, about 10%, about 20%, about 25%, about 50%,about 75%, about 90%, about 95%, about 96%, about 97%, about 98%, about99%, 100% or any range therein of the hot spots or cold spots in apolynucleotide sequence.

The position or reading frame of a hot spot or cold spot is also afactor governing whether SHM mediated mutagenesis that can result in amutation that is silent with regards to the resulting amino acidsequence, or causes conservative, semi-conservative or non conservativechanges at the amino acid level. As discussed below, these designparameters can be manipulated to further enhance the relativesusceptibility or resistance of a nucleotide sequence to SHM. Thus boththe degree of SHM recruitment and the reading frame of the motif areconsidered in the design of SHM susceptible and SHM resistantpolynucleotide sequences.

As used herein, “somatic hypermutation” or “SHM” refers to the mutationof a polynucleotide sequence initiated by, or associated with the actionof activation-induced cytidine deaminase, uracil glycosylase and/orerror prone polymerases on that polynucleotide sequence. The term isintended to include mutagenesis that occurs as a consequence of theerror prone repair of the initial lesion, including mutagenesis mediatedby the mismatch repair machinery and related enzymes.

As used herein, the term “UDG” refers to uracil DNA glycosylase, one ofseveral DNA glycosylases that recognize different damaged DNA bases andremove them before replication of the genome. Typically, DNAglycosylases remove DNA bases that are cytotoxic or cause DNA polymeraseto introduce errors, and are part of the base excision repair pathwayfor DNA. Uracil DNA glycosylase recognizes uracil in DNA, a product ofcytidine deamination, leading to its removal and potential replacementwith a new base.

The term “pol eta” (also called PolH, RAD30A, XPV, XP-V) refers to alow-fidelity DNA polymerase that plays a role in relication throughlesions, for instance, replication through UV-induced thymidine dimers.The gene for pol eta is defective in Xeroderma pigmentosum variant typeprotein, XPV. On non-damaged DNA, pol eta misincorporates incorrectnucleotides at a rate of approximately 3 per 100 bp, and is especiallyerror-prone when replicating through templates containing WAdinucleotides (W=A or T) (Gearhart and Wood, 2001). Pol eta has beenshown to play an important role as an A/T mutator during SHM inimmunoglobulin variable genes (Zeng et al., 2001). Representativeexamples of pol eta include without limitation, human (GenBank AccessionNo. BAA81666), rat (GenBank Accession No. XP_(—)001066743), chicken(GenBank Accession No. NP 001001304), canine (GenBank Accession No.XP_(—)532150) and mouse (GenBank Accession No. NP_(—)109640) forms.

The term “pol theta” (also called PolQ) refers to a low-fidelity DNApolymerase that may play a role in crosslink repair (Gearhart and Wood,Nature Rev Immunol 1: 187-192 (2001)) and contains an intrinsicATPase-helicase domain (Kawamura et al., Int. J. Cancer 109(1):9-16(2004)). The polymerase is able to efficiently replicate through an abasic site by functioning both as a mispair inserter and as a mispairextender (Zan et al., EMBO Journal 24, 3757-3769 (2005)). Representativeexamples of pol theta include without limitation, human (GenBankAccession No. NP_(—)955452), rat (GenBank Accession No. XP_(—)221423),chicken (GenBank Accession No. XP_(—)416549), canine (GenBank AccessionNo. XP_(—)545125), and mouse (GenBank Accession No. NP_(—)084253) forms.Pol ete and Pol theta are sometimes referred to collectively as “errorprone polymerases.”

Phage Display Terminology

“Phage display” is a technique by which variant polypeptides aredisplayed as fusion proteins to at least a portion of a coat protein onthe surface of phage, e.g., filamentous phage, particles. A utility ofphage display lies in the fact that large libraries of randomizedprotein variants can be rapidly and efficiently sorted for thosesequences that bind to a target molecule with high affinity. Display ofpeptide and protein libraries on phage has been used for screeningmillions of polypeptides for ones with specific binding properties.Polyvalent phage display methods have been used for displaying smallrandom peptides and small proteins through fusions to either gene III orgene VIII of filamentous phage. Wells and Lowman, Curr. Opin. Struct.Biol., 3:355-362 (1992), and references cited therein. In monovalentphage display, a protein or peptide library is fused to a gene III or aportion thereof, and expressed at low levels in the presence of wildtype gene III protein so that phage particles display one copy or noneof the fusion proteins. Avidity effects are reduced relative topolyvalent phage so that sorting is on the basis of intrinsic ligandaffinity, and phagemid vectors are used, which simplify DNAmanipulations. Lowman and Wells, Methods: A companion to Methods inEnzymology, 3:205-0216 (1991).

A “phagemid” is a plasmid vector having a bacterial origin ofreplication, e.g., Co1E1, and a copy of an intergenic region of abacteriophage. Phagemids may be used on any known bacteriophage,including filamentous bacteriophage and lambdoid bacteriophage.Generally, the plasmid will also contain a selectable marker forantibiotic resistance. Segments of DNA cloned into these vectors can bepropagated as plasmids. When cells harboring these vectors are providedwith all genes necessary for the production of phage particles, the modeof replication of the plasmid changes to rolling circle replication togenerate copies of one strand of the plasmid DNA and package phageparticles. The phagemid may form infectious or non-infectious phageparticles. This term includes phagemids, which contain a phage coatprotein gene or fragment thereof linked to a heterologous polypeptidegene as a gene fusion such that the heterologous polypeptide isdisplayed on the surface of the phage particle.

The term “phage vector” means a double stranded replicative form of abacteriophage containing a heterologous gene and capable of replication.The phage vector has a phage origin of replication allowing phagereplication and phage particle formation. The phage is preferably afilamentous bacteriophage, such as an M13, f1, fd, Pf3 phage or aderivative thereof, or a lambdoid phage, such as lambda, 21, phi80,phi81, 82, 424, 434, etc., or a derivative thereof.

The term “coat protein” means a protein, at least a portion of which ispresent on the surface of the virus particle. From a functionalperspective, a coat protein is any protein, which associates with avirus particle during the viral assembly process in a host cell, andremains associated with the assembled virus until it infects anotherhost cell. The coat protein may be the major coat protein or may be aminor coat protein. A “major” coat protein is generally a coat proteinwhich is present in the viral coat at preferably at least about 5, morepreferably at least about 7, even more preferably at least about 10copies of the protein or more. A major coat protein may be present intens, hundreds or even thousands of copies per virion. An example of amajor coat protein is the p8 protein of filamentous phage.

A “fusion protein” and a “fusion polypeptide” refer to a polypeptidehaving two portions covalently linked together, where each of theportions is a polypeptide having a different property. The property maybe a biological property, such as activity in vitro or in vivo. Theproperty may also be a simple chemical or physical property, such asbinding to a target molecule, catalysis of a reaction, etc. The twoportions may be linked directly by a single peptide bond or through apeptide linker containing one or more amino acid residues. Generally,the two portions and the linker will be in reading frame with eachother.

II. Introduction to Somatic Hypermutation (SHM)

Natural mechanisms for generating antibody diversification have evolvedutilizing the process of somatic hypermutation (SHM), which triggersdiversification of the variable region of immunoglobulin genes,generating the secondary antibody repertoire thereby allowing affinitymaturation of a humoral response. Thus, by directing hypermutation todefined hypervariable regions of an immunoglobulin (Ig) protein scaffoldand applying selective pressure to identify improved antibodies, theimmune system has developed a diversification strategy capable ofrapidly evolving high affinity antibodies within about three weeks inresponse to antigen exposure.

AID is expressed within activated B cells and is an essential proteinfactor for SHM, as well as class switch recombination and geneconversion (Muramatsu et al., 2000; Revy et al., 2000). AID belongs to afamily of enzymes, the APOBEC family, which share certain features withthe metabolic cytidine deaminases but differs from them in that AIDdeaminates nucleotides within single stranded polynucleotides, andcannot utilize free nucleotide as a substrate. Other enzymes of theAID/APOBEC family can also act to deaminate cytidine on single strandedRNA or DNA (Conticello et al., (2005)).

The human AID protein comprises 198 amino acids and has a predictedmolecular weight of 24 kDa. The human AID gene is located at locus12p13, close to APOBEC-1. The AID protein has a cytidine/deoxycytidinedeaminase motif, is dependent on zinc, and can be inhibited bytetrahydrouridine (THU) which is a specific inhibitor of cytidinedeaminases.

Even prior to the discovery of AID, it was noted that SHM occurs morefrequently in cytidines that are within the context of WRCY (AT/GA/C/AT)motifs. There is now accumulating evidence that this motif for SHMlikely represents a composite of this hot spot motif for AID deaminationand for initiating error prone repair by the DNA polymerases pol eta andpol theta (Rogozin et al. (2004); Zan et al. (2005)).

High levels of DNA transcription have been shown necessary but alone arenot sufficient for AID mediated mutagenesis. In vivo, SHM begins about80 to about 100 nucleotides from the transcription start site, butdecreases in frequency as a function of distance from the promoter. AIDhas been shown in vitro to interact directly with the transcriptionalelongation complex, but not the transcriptional initiation complex, andthis interaction may be dependent upon the dissociation of theinitiation factors, that occurs as the transcriptional initiationcomplex converts to the fully processive, elongation-competenttranscription elongation complex (Besmer et al., 2006).

Since AID is only able to deaminate cytidines on single stranded DNA, itis likely that the requirement for transcription reflects the generationof single stranded regions by transcription bubbles. Studies withpurified AID in vitro however suggest that AID binding is sequenceindependent, potentially allowing a scanning mode for hot spot capturethat is driven by active transcription of the gene. In vitro studiessuggest that AID has an apparent Kd for single stranded DNA in the rangeof 0.3 to 2 nM, and that the complex has a half life of 4-8 minutes. Theturnover number of purified AID on single stranded DNA is approximatelyone deamination every 4 minutes, (Larijani et al., (2006)).

AID acts on DNA to deaminate cytidine residues to uracil residues oneither strand of the transcribed DNA molecule. If the initial (C→U)lesion is not further modified prior to, or during DNA replication thenan adenosine (A) can be inserted opposite the U nucleotide, ultimatelyresulting in C→T or G→A transition mutations. The significance of thischange at the amino acid level depends upon the location of thenucleotide within the codon within the reading frame. If this mutationoccurs in the first or second position of the codon, the result islikely to be a non conservative amino acid substitution. By contrast, ifthe change occurs at the third position of the codon reading frame,within the wobble position, the practical effect of the mutation at theamino level will be slight because the effect of the nucleotide changewill be silent or result in a conservative amino acid substitution.

Alternatively, the C→U lesion, and potentially the neighboring bases canbe acted upon by DNA repair machinery, which in SHM, leads to repair inan error prone fashion. Studies in knock out mice have established thatbase excision repair via uracil DNA glycosylase (UDG), plays a role inmediating the mutation of A and T residues close to hot spot motifs;(Shen et al (2006)). Additionally there is increasing evidence that thecreation of abasic sites by UDG recruits error prone polymerases, suchas pol eta and pol theta, and that these polymerases introduceadditional mutations at all base positions in the surrounding sequence(Watanabe et al. (2004); Neuberger et al (2005)). It is believed thatpol eta is central to the creation of A mutations during SHM and isparticularly error prone for coding strand adenosines proceeded by A orT (W/A) that are preferentially mutated to G.

It has been observed that in antibody genes, codon usage and preciseconcomitant hot spot/cold spot targeting of AID activity and pol etaerrors in the CDRs and FRs, respectively, has evolved under selectivepressure to maximize mutations in the variable regions and minimizemutations in the framework regions (Zheng et al., JEM 201(9): 1467-1478(2005)) for example, observed that the precise alignment of C and Gnucleotides within the codons preferentially used within an antibodygene causes most C to T and G to A mutations to be silent orconservative. Juxaposed on the precise placement of Cs and Gs, Zheng etal., also observed the preferential placement of As and Ts in hot spotsof pol eta in the variable regions and the exclusion from these sites inthe framework regions.

The regulation of SHM in vivo and the determinants that direct and limitSHM to the Ig locus has been the subject of intense debate andexperimental research. The rate of SHM observed in vivo has been shownto be at least partially dependent upon, for example, the followingfactors: 1) the AID expression levels and AID activity levels within aparticular cell type; (Martin et al. (2002), Rucci et al., (2006)), 2)the degree of AID post translational modification and degree of nuclearlocalization; (McBride et al. (2006), Pasqualucci et al. (2006), Muto etal. (2006)), 3) the presence of immune locus specific enhancer regions,E-box motifs, or associated cis acting binding factors; (Komori et al.(2006), Schoetz, et al. (2006)), 4) the proximity of the targetedsequence to the transcriptional initiation site/promoter region; (Radaet al., (2001)), 5) the rate of transcription of the target sequence;(Storb et al., (2001)),6) the degree of target gene methylation;(Larijani et al (2005)), 7) the genomic context of the target gene, ifintegrated into the cell's genomic DNA; 8) the presence or absence ofauxiliary factors, such as Pol Eta, MSH2; (Shen et al. (2006)), 9) theexistence of hotspot or coldspot sequences within the target sequence;(Zheng et al. (2005)), 10) the existence of inhibitory factors;(Santa-Marta, et al. (2006)), 11) rate of DNA repair within the celltype of interest, (Poltoratsky (2006)), 12) the formation of local DNAor RNA hairpins structures; (Steele et al. (2006)), and 13) thephosphorylation state of histone H₂B (Odegard et al. (2005)).

III. Polynucleotides for Somatic Hypermutation

The degree to which a polynucleotide sequence or motif is a SHM “hotspot” or “cold spot” is derived from a statistical analysis of SHMmutations identified in antibody sequences, as described in priorityU.S. application No. 60/902,414, and is shown in Tables 2 and 3 below.These Tables show the 3-mer, 4-mer, and 6-mer motifs ranked by z-scorefor their ability to attract SHM-mediated mutation.

TABLE 2 3- 3-mer 4-mer 4-mer 4-mer 4-mer mer z-score 4-mer z-score 4-merz-score 4-mer z-score 4-mer z-score ATA 271.09 AATA 249.23 TACC 92.73ACGA 19.69 CTGG −55.05 AGC 185.10 AGCA 225.50 GAAA 89.97 TTTT 17.21 CGGA−56.07 TAT 178.79 ATAT 224.06 CTGC 88.23 TTCT 16.95 ACGG −58.65 CAG176.52 AACA 215.78 CCAA 87.55 GATC 16.55 GCCT −61.62 ACA 161.58 ATAA213.14 TATC 86.83 TGTA 15.70 CGCC −62.50 CCA 156.43 ATCA 193.93 CCCA86.81 CCCC 14.29 CTTG −63.02 ATT 128.07 TACA 190.78 GCTA 84.30 TTCC 8.07AGTG −64.08 AAT 123.91 CACA 183.94 CTTA 83.60 CGCA 7.95 GGAC −66.33 CAC113.31 ACAA 182.20 GCAA 83.41 CCTG 6.44 CCCG −68.14 CAT 106.72 ATTA174.57 ATCC 82.88 AAGT 6.21 GTGA −69.31 GCT 99.04 CAGA 172.86 GAAT 82.09GTTA 5.83 TTGT −70.87 TCA 92.35 AACT 171.38 ATTC 80.57 GTAA 5.54 GCGA−71.78 TAC 90.32 AGAT 167.36 AGCC 79.90 GACT 5.46 GTTT −73.35 ACT 84.63ACAG 165.72 CTCA 78.97 TCCT 4.16 GGGA −75.77 ATC 82.30 CAAC 163.72 CCAG78.46 GACC 2.64 CGTA −76.30 AGA 78.69 TATA 159.43 AGTA 78.05 GGAT −0.62TCGA −76.40 CTA 71.32 ATAC 157.31 TAGC 76.80 TCTG −1.62 CGAG −78.05 GCA70.80 ACTA 152.17 ATTT 74.50 GCTG −2.06 AGGG −81.46 GAT 68.06 CAGC148.78 ACTG 74.10 GATG −2.19 GAGT −82.94 CTG 67.83 ACCA 146.54 TCAC71.95 ACCG −2.66 CCGG −85.06 ACC 65.99 AAGC 145.36 CTGA 68.58 TTTC −4.30GAGG −85.74 GAA 59.03 AGAA 144.62 CCTA 67.05 TAGT −4.65 GTTG −86.35 TGA56.50 AAAA 136.44 TCTA 66.67 CGCT −5.54 TCCG −88.86 ATG 52.18 ACAT135.69 AATG 66.07 AGCG −5.58 GTTC −89.62 CAA 48.79 AGCT 134.58 GCAT65.56 CCCT −7.38 CGGC −90.00 AAA 39.39 CAAT 133.12 ACCC 62.47 CCTC −7.50GCGC −91.60 AAC 37.15 GATA 131.74 TCAT 61.22 TGGA −8.79 CTCG −92.05 TTA35.04 ACAC 130.35 TGCT 61.11 CTGT −10.50 TGGC −92.93 TAA 31.78 ATCT128.86 CTAG 59.03 GTAT −10.53 TCGC −96.14 AAG 24.73 CACC 125.86 ACTT58.98 TATG −13.14 TGTG −96.30 CTT 17.61 CATA 125.75 AGAG 58.81 AAGG−13.25 TTGG −100.73 TTC 16.92 ATAG 121.65 TTAC 57.51 CCGC −13.98 GGTT−102.17 GTA 15.61 TAAT 121.29 TTTA 56.94 ATGG −13.99 GCCG −104.21 TAG13.84 CAAA 121.00 TCAG 56.45 CGAA −14.21 CCGT −105.94 GGA 11.44 TATT120.42 ATGC 54.70 TCTT −15.45 GTCT −108.78 TTT 6.80 CTAA 119.93 AGAC53.01 TGAC −16.19 GGCC −110.06 AGT 2.60 CATC 118.61 TGAT 51.51 CCTT−16.61 GACG −112.93 CTC −1.47 TTCA 117.73 GCAC 51.04 CACG −19.16 TGGT−115.42 TCC −5.22 AAAC 116.35 AGGA 50.16 GGCA −21.99 GTGC −117.74 CCT−5.42 TTAT 114.64 TAAG 49.76 TCCC −23.02 TTCG −118.98 CCC −7.09 AAAT114.43 CAGT 49.09 AACG −26.20 ACGT −121.92 GAG −8.26 CCAT 113.51 ACTC46.69 CGAT −27.41 GCGG −124.24 TGC −14.70 ACCT 111.92 AGTT 45.47 AGGT−29.09 TGCG −126.58 TCT −18.88 TAAC 111.26 CAAG 43.20 TCTC −29.53 TGGG−127.63 GAC −23.11 CTAT 110.83 CTCC 43.07 TTGC −29.86 GTCC −128.75 AGG−27.85 TAAA 110.30 GTAC 42.84 CCGA −32.32 GGGC −132.40 GCC −38.10 CCAC110.05 GAAC 42.62 TGAG −34.69 GGGG −133.41 TGG −40.97 AATT 109.92 GAGC41.24 ATGT −34.90 TCGT −135.34 TTG −43.86 TGCA 107.12 GCCA 40.88 TAGG−37.28 GGTG −135.80 ACG −61.29 CATT 106.83 GCTT 39.88 GGCT −38.30 CGTT−136.77 GTT −62.25 TCAA 104.12 CAGG 37.16 GCCC −40.66 TGTC −137.57 CGA−62.60 AAAG 103.76 GATT 35.99 GGAG −44.01 GTGT −142.24 TGT −64.56 TACT101.53 GACA 35.71 TGTT −44.49 CGGT −144.04 GGC −70.30 AAGA 100.90 CTTC34.67 CGAC −45.06 GTGG −149.24 CGC −82.93 CACT 100.32 CTCT 33.87 GGTA−46.07 CGTC −155.95 CCG −85.43 AACC 99.86 GAAG 31.97 AGGC −46.08 GGTC−158.84 GGG −97.46 GCAG 99.17 TTGA 31.29 TACG −46.78 TCGG −159.56 GTG−110.90 ATGA 98.38 CTTT 28.94 AGTC −46.82 CGGG −159.99 GGT −112.41 CTAC95.93 TTAG 27.86 ACGC −47.10 GGGT −162.17 CGG −116.32 TCCA 95.63 GGAA26.38 ATCG −48.15 GGCG −171.27 GCG −118.80 AATC 95.61 ATTG 25.55 GTCA−52.15 CGCG −172.40 TCG −125.83 TGAA 93.81 CATG 24.39 TTTG −52.48 CGTG−180.34 GTC −126.67 TTAA 93.67 GCTC 22.00 GTAG −53.73 GCGT −194.57 CGT−130.10 TAGA 93.03 GAGA 21.55 TGCC −54.56 GTCG −207.74

TABLE 3 6-mer z- 6-mer score ACAGCT 266.45 ATTAAT 248.7 ATAATA 227CAGCTA 223.27 AATATA 220.6 AATACA 215.65 AGCTAC 211.24 AGATAT 211.07AGCTAA 210.24 ATATAT 209.3 AATACT 203.19 ATATAC 192.44 ATAACT 190.78ATATTA 189.76 ATAGCA 186.89 ATACCA 186.58 ATACAA 181.41 GCAGCT 180.69ATTACA 180.46 CAGCTC 180.29 ATAGCT 180.08 AATAAT 179.41 AGCTAT 178.14CAGCTT 176.31 ATATCT 174.41 AGCTGC 169 CAGCTG 167.78 AGCTGA 167.41AATAAA 167.35 ACTACA 167.11 AACAGC 167.08 ATTATT 166.89 AAGCTA 166.44ACTACT 164.71 AATACC 164.29 TATTAT 164.1 ACAGCA 161.72 AGCAGA 160.66AGCAAT 159.61 TAATAC 159.28 AATCCA 156.67 AATAGA 156.3 TATACA 155.5AGCTCC 153.55 CATATA 152.22 ATACAT 151.77 TATATT 150.71 TAATAT 150.37ATTACT 150.2 TCAGCT 149.79 AACTAC 149.11 AAAGCT 148.88 CAGCAT 147.47ATACAC 147.42 ATAGAT 147.33 ATCAGC 147.06 AGATAC 146.34 AGCACA 146.01CAGATA 145.75 TAGCTA 145.22 TTAGCT 144.8 AAGCTG 143.55 CACAGC 141.38ACAACT 140.89 CATACA 139.87 AGCAGC 139.64 ACTATT 139.36 CCAGCT 137.43GATACA 136.87 AGCTTC 136.64 AGCTCA 136.52 ACCAGC 136.02 AAATAC 135.35AGCTTA 135.22 AGAGCT 134.71 TAACTA 134.57 TACTAC 134.52 AACTAT 133.79ATAAAC 132.79 TAGATA 132.74 AACACA 131.7 CTAATA 131.46 AATAGC 130.99GAGCTA 130.78 ATACTA 130.56 ATATCA 130.47 CTACTA 130.24 ATACAG 129.95CCAGCA 129.73 CAGCAG 129.37 AATGCA 128.88 ACTAAT 128.87 AGCTTT 128.11ATCCAC 128.11 GAAGCT 126.98 CAGCAA 126.51 ACCACC 126.44 GCTACA 126.36AGCTGT 126.35 ATAACA 126.34 AGTTAT 125.56 TTACTA 125.4 AATTAC 124.76AATTCA 123.97 CAGCAC 123.54 ACAGCC 123.25 TTAATA 122.8 AGTATT 122.69CAACTA 122.15 CAATAA 121.87 AGCAAC 121.8 ATCTAC 121.63 TACACC 121.61AGCACC 121.59 ATAGCC 120.05 TAGCTG 119.3 AAAACA 119.25 ATTATA 119.17AGTACT 118.38 CACCAT 117.87 ATCTAT 116.19 ACCATT 115.23 TACTAT 115.17TCAGCA 115.13 AGCATA 114.84 TATTAA 114.69 CAAGCT 113.83 AGATGA 113.27GATATA 112.88 TAGCTT 112.54 TATTAC 111.72 AGCTCT 111.46 TCACCA 111.34ATAGTA 110.66 ATACCT 110.48 AGCATC 109.68 TATCTA 109.46 TACAAC 108.83GCAGCA 108.59 AGTAAT 108.57 TGCACA 108.53 TTTATT 108.51 ATGATA 108.34CAAATA 108.12 ACAATA 107.6 AATAGT 107.19 AACAAC 107.08 CACCAG 107.01TAGCTC 106.68 TACAGC 106.65 AACTGA 106.63 GCATAT 106.63 GAGCTG 106.39ATTCAC 106.22 AAATAA 105.92 TAGCAA 105.71 CCAGAT 105.22 ACCATC 105.14AATAAC 105.1 TACCAT 104.92 AGAACA 104.85 ATCATA 104.56 ATCACC 104.5AGAAAT 104.29 ATATAA 104.19 CATATC 103.97 ATTCCA 103.78 GGAGCT 102.99TACAGA 102.58 TACTAA 102.18 ATCACT 102.01 ATATGA 101.89 AAACAG 101.82ACACAG 101.77 ACACCA 101.38 ACAACC 101.23 TAAGCT 100.84 CAATAG 100.69CTATTA 100.61 TTACCA 100.56 AGTACA 100.42 AACCAC 100.39 CCACCA 100.19AAACAC 99.94 ATAAAT 99.38 GCTATA 99.35 GTAGCT 99.14 CAGCCA 99.11 TTCAGC99 AGACAC 98.97 AGCACT 98.85 CCAATA 98.8 AAACCA 98.68 CAGCCT 98.34AAGCAC 98.34 ACTGCA 98.25 AGAAGC 98.23 CCATCA 98.1 CAACCA 97.53 CAACTG97.51 ATTAGC 97.37 AATATT 96.98 ACCACA 96.82 ATATGC 96.53 GTATTA 96.49CATAGC 96.33 GTATAT 96.2 ACCAAC 96.14 CAGATC 96.05 AACATA 96.05 AGATCC95.89 CTACCA 95.82 GATCCA 95.8 ATTGCT 95.61 ACCATA 95.61 CATCTA 95.61CCAGCC 95.4 ACCTAC 95.39 TCAACT 95.32 ATGCAC 95.22 GAAATA 95.07 TATAGC94.95 TACCAC 94.81 AGCTAG 94.59 CCATAT 94.32 TATATA 94.2 CATATT 94.16TAATAA 94.05 AGAACT 93.81 TATCAC 93.66 CACCAC 93.38 AAAGCC 93.36 CTACAG93.16 GCAGAT 93.16 AGATCA 93.03 ACTTCA 92.78 ACACAC 91.91 ACCACT 91.48AAGCTT 91.27 ACCAAT 90.89 CTAGCT 90.83 ATTTAT 90.72 CAGTTA 90.71 CATAGA90.61 ATACTG 90.19 ATTACC 90 TATCAT 89.91 ACTATA 89.16 TACACA 89.01GCTGAA 88.67 CCATTA 88.62 TGCTAT 88.19 TACATA 88.12 CACCAA 88.08 ATAGTT87.88 CACCTA 87.77 GCACCA 87.64 CTATCA 87.58 GCTATT 87.58 TATTAG 87.34CCACCT 87.28 AGAACC 87.26 ACTACC 87.25 TATAAT 87.06 ATTTCA 86.86 TAGCAG86.76 AAGCTC 86.67 AACCAA 86.61 AATATC 86.37 TAGTAA 86.29 GCTGAT 86.25TATATC 86.21 TAATTA 86.14 AACCAT 86.06 ATAGAC 86.03 CCATCT 85.84 TTATTA85.75 TCAGCC 85.73 ACATAC 85.65 ACATAG 85.6 CACAAT 85.55 GTAATA 85.54GAAGCA 85.45 TCATAT 85.24 CAGCCC 85.03 ACCTAT 84.68 AGCCAC 84.68 CAGTAA84.62 CCAACA 84.17 AAAAGC 84.12 AACTGC 83.95 CCAACT 83.78 ATCATT 83.47AGAGCA 83.38 GATACT 83.35 CCACAG 83.35 ATAATT 83.26 TAAACA 83.21 ACATAT82.99 GCTACT 82.86 CAGTAT 82.76 ATCACA 82.36 TCAACA 82.34 AGCCCA 82.25AATTAT 82.21 ATCATC 82.17 TGCTAC 81.84 GCTTCA 81.55 CCACTA 81.49 GCTGCA81.44 TAGTTA 80.97 AATCAA 80.92 CAATTA 80.84 CTGCTA 80.71 ATATAG 80.66TGCACC 80.52 AAGACA 80.5 TAATAG 80.31 TGCAGC 80.23 CCTCCA 80.17 GATGCA80.15 AACTCC 80.09 TCCAGC 80.02 ACACTG 79.79 TATAAC 79.77 TTATAA 79.58CAACAA 79.5 GCTAAT 79.35 TGATAC 79 AGATCT 78.63 ATAACC 78.57 AGAAAC 78.2ATTGCA 78.18 AACACC 78.06 TGCATT 78 CAACTC 77.9 GTACTA 77.86 ACTCCA77.83 CAGATG 77.71 TGCAGA 77.69 AAGAAA 77.67 TCCACC 77.66 TAACCA 77.39TAACAG 77.34 TTATAT 77.04 TCTATT 76.92 ACACTA 76.75 CACTAA 76.68 GTAGCA76.59 AGCCAT 76.52 TCATCT 76.5 CACTAT 76.28 CAATAT 76.05 CACAGA 76.03AGTTAC 75.97 ATACTC 75.91 TATATG 75.77 CACTAC 75.68 ATTTCT 75.56 TACCAA75.44 GCAATA 75.24 ATCTCA 74.72 ACAGAT 74.63 TCACCT 74.58 CATCAG 74.49TCAGAT 74.33 AGTAAC 74.08 CTACAC 73.7 AATGAT 73.53 ATTAGT 73.5 TAGTAC73.49 TAACTG 73.35 AAAATA 73.29 AAAACT 73.19 ATTTAC 72.97 ATCTGA 72.97ATCCAT 72.95 ATACCC 72.75 AACTTC 72.62 AATACG 72.39 AAATCA 72.22 TTCACA72.18 CAGATT 72.08 CAGAAA 71.97 ACACAT 71.91 AAGATA 71.91 CTGCAG 71.63GCAACT 71.57 GATATT 71.57 AGATTC 71.53 ACCAGA 71.47 CTATAT 71.38 TGATAT71.06 AAGAGC 70.89 ATACGC 70.65 CTGATA 70.47 GATAAA 70.39 ACATCC 70.36AAACTA 70.26 ATCAAT 70.13 GAAACA 70.11 CATCAT 70.01 AGCTTG 70.01 TGAGCT69.96 CTATAA 69.96 ATTCAT 69.85 TACTGC 69.83 CAGAGA 69.69 CATTTA 69.68AGCTGG 69.06 GAATCA 68.99 TTATTT 68.98 ATCTGC 68.96 TAGCAC 68.84 ATGCTA68.58 TATACT 68.54 TCATCA 68.5 AGATGC 68.48 ATAGCG 68.46 CATACT 68.15TAGCAT 68.15 TACAAA 68.02 TACCTA 67.99 CATCTT 67.88 ATCAAC 67.83 ACCTTC67.82 TTAGCA 67.82 AGTAGC 67.72 TTGCTA 67.61 TAAGCA 67.57 AATATG 67.49TCACTA 67.42 CATTAA 67.2 AGCAAA 67.17 GGCTAT 67.15 ATGCAA 67.06 ACACCC67.05 GCAGTA 67.04 AGTAAA 67 TTCACC 66.71 GATACC 66.69 CTACAA 66.54CTGAAA 66.27 ATGTAT 66.24 CACCTT 66.08 ACCCAG 65.77 ATATCC 65.64 CAAAGC65.58 ACAGTA 65.5 CATACC 65.47 TGAATT 65.43 TATTCA 65.2 GATATC 65.15ACAAAT 65.04 CCATTT 64.91 AAAAAC 64.81 GCTCCA 64.64 AAGCCA 64.61 CCTTCA64.45 GAGCTT 64.45 ATAGAA 64.31 TGAAGC 64.22 GAACCA 64.2 ACAGAC 64.16ACAGAG 64.14 TGTATA 64 TGAACC 63.94 TTATCA 63.94 AACAGA 63.94 GATTCA63.93 ATGAAT 63.83 GCTGCT 63.71 CACACA 63.58 GCAGCC 63.54 TAGCCA 63.4GAGCTC 63.35 AACTCA 63.19 GTATCA 63.01 CATAAT 62.96 TCCACA 62.68 CAGAAG62.65 CCCAGC 62.57 CGCTAT 62.55 CCTACT 62.52 CAATAC 62.45 CAACTT 62.28AGAATC 62.21 GAGCAC 62.17 TCTGCA 62.09 CAATCC 61.99 AGAATT 61.72 CATTAC61.65 ACTGCT 61.63 AACACT 61.62 GTAACA 61.62 TATCAG 61.58 ATGAAC 61.56CAACAT 61.55 TCAATA 61.47 TGCATC 61.37 GCACAG 61.24 AGAGCC 61.12 AGTATA61.1 GTAGAT 60.86 TACACT 60.8 TATCCA 60.75 AGCATT 60.65 ATTAAA 60.65ACAAGC 60.61 ACTGAT 60.54 CAACAG 60.42 ATGCTG 60.37 TATCAA 60.3 AGTTGA60.16 TTTACT 60.02 CTTCAC 59.96 GAAGAT 59.8 CATCTG 59.68 ATCCCA 59.65CAACAC 59.49 AACATC 59.39 AAGCAG 59.37 CATCAC 59.3 ACTAGC 59.24 ACAACA59.21 CATAAC 59.02 TATTTC 58.98 CCATAA 58.89 CACCCT 58.6 ACACCG 58.31TACTAG 58.31 TGAATA 58.12 ACAATC 58.11 AGGAGC 58.09 TGAGCA 57.87 TATGAT57.78 TATACC 57.77 GATATG 57.64 TCTGCT 57.47 AGTAGT 57.38 ACCAAA 57.17TGTAAT 57.16 CAGCGA 57.12 AAGCAT 57.06 GATGCT 57.03 CATTTC 56.98 AAGATG56.93 ATCCAG 56.88 CATATG 56.87 TGGATT 56.83 TGCAAC 56.76 CACCTC 56.75CAGACT 56.73 ATGCAG 56.72 GTAACT 56.7 AGTAGA 56.45 TATGCA 56.42 GGAATA56.3 AGTATC 56.23 CATTAG 56.19 CAGTAC 56.18 TACATC 56.14 AAAGCA 56.13TCTCCA 56.01 ACAGAA 55.96 GGAGCA 55.88 CAGCCG 55.8 CTGCAC 55.6 AGCAGT55.46 CACATA 55.45 TATCTG 55.37 TACTCA 55.36 CTTATA 55.34 GACACA 55.17TGTATT 55.14 GAATCT 55.12 AACAGT 55.1 ATCAGA 55.06 GCATCT 54.8 AACTAA54.79 CAGCGC 54.76 ACACAA 54.74 TAACAA 54.73 TGCATA 54.73 TTACAG 54.68GAAGCC 54.6 AAGAAC 54.37 TTACTG 54.36 GTTTAT 54.25 ACCAGT 54.25 AATCCT54.22 ACAAAG 54.18 TCACAG 54.18 ACTATG 54.15 GATGAT 54.08 TGCAAT 54.03GTAATT 53.95 TTAGTA 53.95 CATGAA 53.93 CATCTC 53.89 AGCCTC 53.8 CACATT53.79 AATTAA 53.78 GCACAT 53.76 ATTGAT 53.75 AAAACC 53.75 TACCAG 53.61ACTAGT 53.57 AAAGAT 53.54 CTCCAA 53.42 CACACT 53.37 CCACAA 53.24 TACAAT53.13 CTATTG 53.01 TAGTAG 52.94 GATCAT 52.84 AATCAT 52.81 ATTCAG 52.71AGTACC 52.64 AAAAAT 52.58 CAGAAC 52.37 ACAGTT 52.35 TGAAAT 52.33 GAGATC52.3 CATTCA 52.24 CGAGCT 52.22 GATAGC 52.17 TCATTA 52.11 CTCCAG 52.03CAGAGC 51.98 TGCTGA 51.92 CCAAGA 51.92 ATAAGC 51.86 TTACAC 51.85 AGATGG51.72 TCTACT 51.69 TTACAA 51.68 TGCAAA 51.62 TAGTAT 51.42 TTTATC 51.26CCCAGA 51.25 GACTAC 51.19 ATTCTA 51.19 CAAAAA 51.15 ATACTT 51.15 ATACGA51.08 ATCTTC 51.06 ACATCA 51.04 AACCCA 51 CATAAA 50.95 TGAAGA 50.88TAGATG 50.83 CTGCAT 50.78 CAAGCA 50.65 AAATCC 50.5 GAACTA 50.47 CTATGA50.36 ACTTAT 50.3 CCAAAT 50.25 CCTGCA 50.24 TACTCC 50.15 GAGCAG 50.07TACCCA 50.02 ACCTCC 49.97 GTTATA 49.88 CATCAA 49.87 TGATAA 49.86 AATCAC49.84 ATTAGA 49.71 CATCCC 49.63 GTATTT 49.61 ACCTGA 49.59 ACTGAA 49.51CATCCA 49.5 TAACAC 49.46 AGAGAT 49.39 AGCATG 49.33 CAACCC 49.27 ACTTCT49.23 ATGATC 49.2 GATAGA 49.19 GAACAG 48.99 CCAAAA 48.88 GAAACT 48.8GACAGC 48.76 CAATGA 48.7 ACAAGA 48.64 CTCAGA 48.55 AGATAA 48.54 CTAGCA48.43 ATCAAA 48.36 TCTTCA 48.34 GATGAA 48.34 ATCCAA 48.27 AACCAG 48.27CACATC 48.25 TCCAAC 48.16 TAAAGC 48.1 AGACCC 48.09 CAGGAA 48.07 TTAACA48.04 TTATTG 48 CATGGA 47.99 CTTCCA 47.96 CAGTTG 47.94 ATATGG 47.86GTATCT 47.79 CTTCAA 47.73 GAGAAC 47.72 TTCACT 47.71 AAAGAA 47.71 ACACCT47.51 AGTTCA 47.47 ACCTGC 47.45 TATGCT 47.44 TTGTAT 47.43 ACAGGC 47.42TCCATA 47.27 TATTCC 47.17 GGCTGA 47.15 TGCTAA 47.05 ACCCCA 46.96 GTAGTA46.89 ATCCTA 46.79 CGCATA 46.68 AATTCT 46.54 GGATCT 46.23 TTATAG 46.2ACTAAA 46.2 CAGACA 46.2 GTACCA 46.16 CAAAGA 46.13 ACTCCT 46.11 CACAGT46.1 AAACCT 46.05 CGCTGA 46.02 AATGAA 45.98 GTTACT 45.95 TACAAG 45.86AGGAAT 45.81 ACTCAA 45.79 ATGACA 45.7 ACCATG 45.69 CATAGT 45.61 ATATTG45.6 AGGTAT 45.57 CTCAGC 45.54 ATATTC 45.46 CTACTC 45.36 TACAGG 45.33CCTCAG 45.33 CACTGC 45.24 GCACCT 45.13 ACTATC 45.05 CTGCTG 44.96 AGCCTT44.9 GGTATT 44.89 TAAATA 44.79 TTCCAC 44.78 CAAAAG 44.78 TTTCAG 44.77TAATGA 44.74 TTACAT 44.73 AACCCC 44.73 ATGGTA 44.66 CACTGA 44.64 CAAATC44.64 CATGCT 44.62 GCTTCT 44.61 TCCATC 44.59 TCAGTT 44.56 ACTGCC 44.54CTTCAT 44.49 TGCTCA 44.45 TGGAAT 44.41 CTTCAG 44.4 ACATCT 44.4 CACCTG44.39 ATGCAT 44.36 CCAACC 44.33 CATTAT 44.25 CTAGTA 44.22 TACAGT 44.18TACTGA 44.12 CTACTG 44.1 TAGAAT 44.07 ACAGCG 44.06 ATGGAT 44.04 TTCATA43.92 ATAAAA 43.84 ACTCAG 43.83 CTGCAA 43.65 CAGGCT 43.52 TGATAG 43.5AGAGAC 43.5 CCATGA 43.49 CTACTT 43.4 ACATTA 43.36 GAATAG 43.29 GCAGTT43.25 CACAAA 43.25 TGAACT 43.25 TGAGAT 43.21 CACTAG 43.13 CCCCAT 43.06CTAACA 42.92 CCAGTA 42.86 CTCCAT 42.76 CAAGAT 42.74 GAACCC 42.71 CCAGAA42.65 TTCATC 42.62 AACCTG 42.6 AGCCCC 42.52 CCTACA 42.47 GGATAT 42.47TCCACT 42.41 ATTACG 42.39 AAGATC 42.32 AGCCTA 42.29 ACACGG 42.21 CTGAAT42.18 CTATTC 42.04 ACAATG 42.01 TCATAA 42 TGAATC 41.89 ATCAGT 41.74GATTTA 41.74 AATCTG 41.72 GCTGGA 41.71 AGCGAT 41.68 TATTTT 41.67 GAATCC41.64 TTTACC 41.63 AGCAGG 41.62 AAATAT 41.58 ATTATC 41.55 GAGATA 41.47CCAGGA 41.41 TCATAG 41.39 GCTTTT 41.33 ATGACT 41.26 GAACTG 41.19 CTGAAC41.19 GGCTAC 41.14 AGCTCG 41.12 ACCCAC 41.04 CAATCA 41.01 AGCGCA 40.99ACTCCC 40.96 CTCCAC 40.95 AATCTA 40.93 GCATCA 40.9 ATTTTT 40.87 TGAAAA40.84 TCACAT 40.84 ATTCCT 40.83 TTGATA 40.69 CACAAC 40.69 TATTGA 40.61AGGCTG 40.57 AATGCT 40.53 TATTTG 40.53 CAGGTA 40.51 CATGCA 40.5 AAACTG40.46 AACAAA 40.38 CTTTCA 40.38 CAAACT 40.38 TATTTA 40.37 GGAACA 40.37GCCACT 40.35 CGCAGC 40.24 TAAATC 40.2 AGGTAC 40.19 ACTGTA 40.17 GAAGGA40.16 CAGTTC 40.09 TTTTAC 40.04 TGAACA 40 GCTATC 39.99 GCTTTA 39.98ATTAAC 39.98 GAATAT 39.96 CCATCC 39.94 TACCTG 39.93 CAAACC 39.91 CACTTC39.84 TTATAC 39.76 TTGCAT 39.73 CTGTAT 39.67 GAAACC 39.64 AGTGAT 39.53CAAGCC 39.3 AGGATT 39.29 CAGTAG 39.29 AGAATA 39.23 ATGCCA 39.23 GTGATA39.2 AATCCC 39.2 AACAAT 39.16 GAAGAA 39.02 TAACAT 39 CAAACA 38.97 AGGATA38.8 AAATGG 38.8 TTTAAT 38.75 TTTACA 38.66 GACACC 38.6 CTTACT 38.54TAAAAC 38.52 TCAGCG 38.41 TTTGCA 38.37 ACAAAC 38.35 GATCTC 38.32 TGGATC38.23 AAAAAA 38.16 CACGAT 38.16 TTTTCA 38.15 AAACAA 38.11 AATCAG 38.1ATGAGA 38.04 CCAATT 38.03 CTATAC 37.99 AGGACA 37.98 GAACAA 37.98 TCCAAA37.84 TTTCCA 37.82 ACTGGA 37.81 AAGCAA 37.77 ATGAAG 37.77 ACAAGG 37.76AAGCCC 37.72 GCTCCT 37.68 ACACGA 37.64 AGCCGA 37.6 CCAGCG 37.57 ATCCCC37.48 TGTAGC 37.33 AGCCGC 37.29 TCAGAA 37.28 TAAAAA 37.16 GATAAT 37.15TCCTAC 37.13 TACTTC 37.09 GAAATG 36.99 ATATTT 36.91 GAACTC 36.81 CTAATG36.79 AACAGG 36.76 AAGGCT 36.76 TCCAAT 36.72 TATGAC 36.67 ACCTCA 36.63TGATGA 36.62 AAGCCT 36.59 GAGACA 36.59 ATGATT 36.47 CCACCC 36.46 GCAATT36.27 CCCACA 36.26 TACTTA 36.25 TGACCA 36.23 CCATAG 36.13 ATTCCC 36.08CCCACT 36.08 AAACCC 35.99 GAACCT 35.97 GTTATT 35.96 CCATAC 35.9 TTCTAC35.9 ATGAGC 35.85 GATCAG 35.85 TATGAA 35.79 CAAGAA 35.7 TATAAG 35.62ATCTCC 35.59 ACTACG 35.54 GAACAC 35.49 TATTGC 35.48 TAAATG 35.47 ATGAAA35.43 GATCTG 35.38 TATAAA 35.37 ATACGG 35.34 ATTATG 35.3 CAAGGA 35.22AAATAG 35.19 AAGACT 35.13 ACCCCC 35.07 AGATTT 35.05 GAGCAT 35.02 CCCCAA35.02 AAATGC 35 TGATCA 34.95 GAGCCC 34.9 ATCTGG 34.82 AGAAGT 34.81ACTAAC 34.76 TGGAGA 34.73 TAATCA 34.7 CAACCT 34.69 GACCAC 34.64 GTAAAA34.56 TCTACC 34.54 GATTAC 34.54 CCAGTT 34.52 ACCAGG 34.5 GCAACC 34.48ACATTT 34.47 ACTTCC 34.46 AAGTAC 34.43 ACCTTA 34.43 TAATTG 34.26 CACCCA34.26 ATCTTT 34.13 TTAATT 34.07 TTGCAC 34.06 CACCCC 34.06 CATGAT 34.02ATAGGT 33.92 GCTACC 33.92 ATAGAG 33.86 AGTTCT 33.81 TGCTTA 33.8 GCTGTT33.73 AAGAAT 33.68 GATTCT 33.67 ACCGCC 33.57 ACAGGG 33.56 CAAGAC 33.52CCACTG 33.47 AAGTAA 33.38 TGTACT 33.36 CTGAAG 33.36 AGACCT 33.33 ACTAGA33.32 AAATCT 33.23 GCTATG 33.22 TTGATT 33.18 TGCTGC 33.18 AGAAGA 33.16AATGGA 33.11 TTCCCA 33.1 AATGGT 33.08 GTTACA 33.07 TCAGGA 33.04 TACACG32.96 TTACTT 32.93 TAAAGA 32.93 CACTTT 32.87 AACTGG 32.82 CTCACC 32.81ACATGC 32.79 AGCCTG 32.79 TCCCAG 32.78 ACATGG 32.77 CACTTA 32.69 CCCCCA32.63 ATGATG 32.59 GCAGAG 32.58 ACATAA 32.53 AAAGTA 32.47 AAAAGA 32.46GAACAT 32.46 CAATTC 32.4 CCACTT 32.39 GGCTTT 32.37 TTCAAC 32.34 GCTTAT32.32 CAGGAT 32.32 AGCCCT 32.3 CAATGC 32.26 TGTATC 32.2 TGATCT 32.2CTGTTA 32.12 ACAATT 32.12 TATCTT 32.05 ATTCAA 32.04 TTCAAA 32.03 CAGACC31.98 ACATGA 31.9 CTAAGC 31.75 CTAAGA 31.7 ATAAAG 31.69 AACTAG 31.56GTACCT 31.55 AGATAG 31.51 CAAAAT 31.5 GTGAAT 31.48 AGCCAA 31.4 GAGATG31.33 GGAGAA 31.29 AATTGC 31.29 ATGGCT 31.23 GCAAAT 31.22 TAGAAC 31.2ATGGAA 31.19 GATGGA 31.15 CTGCTC 31.09 CCAGAC 31.09 ACTCAT 31.09 CGAACA31.02 AGCCAG 31.01 GGATAC 31.01 GCAGAA 30.98 GTAAAT 30.95 TTTATA 30.85TGCTTC 30.8 CTCAAC 30.7 AAAGAC 30.65 GCTCAA 30.56 ACAGTC 30.55 CACAAG30.53 TGGATA 30.52 GCATAG 30.51 ACCTGG 30.5 CTCCCA 30.43 TGATTC 30.33GCTGTA 30.33 GCATAC 30.26 TCAAGC 30.25 CAGAAT 30.22 TCATAC 30.18 CATCCT30.14 TGAAAC 30.04 AAACTC 30 GCATTT 29.91 AAGGAC 29.86 ACAAAA 29.84GAGTAT 29.79 AAATGA 29.74 AGCGGA 29.72 GAATTA 29.71 AGTGAA 29.7 AACAAG29.69 TCAAGA 29.63 AACCTT 29.53 GAATAA 29.53 CTCACA 29.49 TCACAA 29.46CCCATC 29.46 TGTGCA 29.41 ATTGGA 29.27 ATTGAA 29.23 ATAATG 29.22 CCTTTA29.21 GGAACT 29.21 TTCAGA 29.18 GCAACA 29.12 ATAATC 29.11 CTCATA 29.07GAATAC 29 CTGATC 29 ACCAAG 28.96 CACAGG 28.94 ATTTCC 28.86 GCATAA 28.83TCCCAC 28.82 GAGCAA 28.81 TCCAGA 28.65 TTCCAT 28.63 GGCACA 28.6 TTTCTT28.55 TAAACC 28.53 AAATTA 28.46 CTTGCA 28.46 ACCTCT 28.41 TCAGTA 28.39GAAGTT 28.37 TACATT 28.33 GACCCA 28.32 GACCAT 28.29 CCACAT 28.23 CATTTT28.22 ATCGCT 28.15 AAGGAA 28.11 TATAGT 27.92 TAACTT 27.89 CTTAGC 27.87CTTAAG 27.83 CCTGCT 27.78 GATACG 27.7 TAGACA 27.69 GGTTCA 27.68 ATGCTT27.68 TTCATT 27.66 TAATCC 27.62 ATATGT 27.59 CCACGA 27.56 AAAATC 27.56GAAGTA 27.52 TGCTCC 27.5 CCCATA 27.47 TTAACC 27.43 TAGAGC 27.38 AGGCTA27.34 GCAAAA 27.32 GCTCAT 27.31 AGGACC 27.3 AGACTA 27.27 CCATTC 27.24ACGACT 27.24 AGGAAA 27.08 TTCCAG 27 TCACCC 26.94 AAATTC 26.9 AACTGT26.84 TTCTAT 26.75 TAATGG 26.71 ACAACG 26.66 AGGTGA 26.64 AGGAAC 26.59TGGTAT 26.57 AAACAT 26.53 AGTTGC 26.52 CAGTGA 26.47 GATTCC 26.47 AGCGAC26.44 ATCAAG 26.44 ACCCCT 26.4 CCCCAG 26.4 CGTATT 26.39 TACTTT 26.39AGACAG 26.37 TTATGA 26.36 CAAGAG 26.32 TGCAGT 26.31 AGGAGA 26.3 CCATGC26.27 GAAAGC 26.23 ACGATA 26.23 CAAGGC 26.22 CTTTAT 26.22 CATTCC 26.22GAAAAT 26.2 CATTGC 26.13 TATACG 26.08 GTAGAA 26.03 GGACCA 26.02 GCTCTT25.97 TGTTAC 25.87 TCCCCA 25.78 TCCATT 25.78 AGAAAA 25.72 CCCAAG 25.69GTGCAT 25.62 TTTTAT 25.58 ACCTTT 25.53 CTACGA 25.52 CCTTAA 25.52 GGCATA25.52 GAAAAC 25.47 AGTTTT 25.42 GAATTC 25.36 GATCAC 25.35 CACACC 25.27AAGCCG 25.26 ACTGAG 25.25 ATCTAA 25.24 AGACTG 25.18 AAGTTA 25.15 TCACTG25.11 ATCGCA 25.08 CGATAT 25.02 GTCATA 24.99 AACCCT 24.98 TTAATG 24.97ACTTTT 24.96 ACGCAG 24.82 ATTTAA 24.79 TGATTT 24.76 CTGATG 24.75 ATCTTA24.75 TATGTA 24.71 GAAGAC 24.69 TTACCT 24.69 TAGATT 24.68 ATAAGT 24.67CGGATA 24.54 CTTTTA 24.43 ACCACG 24.41 ACAGGA 24.4 TATGGA 24.4 TTACTC24.37 GCAAAG 24.34 GAGGCT 24.32 ATCATG 24.24 TGTTAT 24.2 GCAAGA 24.19CTGGAA 24.11 CTATTT 24.06 TCCATG 24.06 AGTGCT 24.05 AGCGCC 24.04 CTGTAA24.03 GAGCCT 24.03 ACCCAT 24.03 TGGAGC 23.99 ATGGAC 23.95 CAGCGG 23.91TAAGAA 23.9 GCATTA 23.88 AGTCAT 23.86 GGAACC 23.86 CCCTCA 23.86 AACCTA23.83 CTTACA 23.77 GGTAAT 23.77 GGAGCC 23.69 CCCACC 23.65 GGAGAT 23.63GTAGTT 23.62 CTGAGC 23.61 TTTCAC 23.61 CTGAGA 23.59 CATAGG 23.58 TTTCAT23.55 AAGTAT 23.48 AATTCC 23.45 TACATG 23.39 GGAAAT 23.35 TGACCT 23.35CGCACA 23.34 TACGAC 23.32 ATTTTC 23.32 CCTGAA 23.3 ACAGTG 23.28 AATCGA23.28 ATCTCT 23.2 GACATG 23.19 AAGTAG 23.18 ATACCG 23.16 GGCAGC 23.07TCTACA 23.02 CTAAAA 23 ACACGC 23 ACCCTG 22.98 TGAAAG 22.87 CACATG 22.71CCTGTA 22.67 TGGTAA 22.66 CAGAGT 22.64 CCGCTA 22.64 GGAATC 22.63 TTCAAT22.52 CTGCTT 22.49 CCTATT 22.49 GGTGCA 22.48 CAGGAG 22.48 CCCCAC 22.46AGGCTC 22.43 CTAACT 22.4 CCAAGC 22.4 GCAGAC 22.36 CCAGGT 22.36 ACTGTT22.3 ACCCTC 22.25 CTATGC 22.23 TCTAAT 22.15 TGGAAA 22.14 CAGTTT 22.08TAATTC 22.08 TCACTT 22.06 TTTTTA 22.01 CCTTCC 21.92 ATCGAT 21.89 AAAATG21.87 GCACAA 21.78 TGCACT 21.71 AAGACC 21.69 AATTGA 21.68 GCATCC 21.65CACTGT 21.65 GAAAAA 21.64 GCTCAG 21.6 AACACG 21.59 GTTGCA 21.57 GCCCCA21.54 GACTAT 21.53 GACCAG 21.52 GTTCAT 21.39 GAGAAT 21.24 TAAAAG 21.2GAATTT 21.15 CACCGT 21.13 GATTAT 21.11 TTTCAA 21.05 ATCCTC 21.03 CTGGAT21 CCTATA 20.97 ATAGGA 20.97 TAGGTA 20.96 GGATTT 20.93 ACTCAC 20.88CGACTA 20.85 GGATCA 20.8 CTACCC 20.78 ACTTAC 20.74 GATAAC 20.71 GATCCC20.66 TACGCA 20.62 GCCACC 20.56 AGACTC 20.56 GACTCA 20.5 CCTTAT 20.39TAGGAT 20.38 AACATT 20.37 ATGCTC 20.32 ACTCTA 20.3 CTGCCA 20.29 TGGCTA20.29 AGTCCA 20.26 CAGTCA 20.24 TTCCAA 20.24 GACATA 20.22 TCTATC 20.15TCCTGA 20.13 ATGGCA 20.05 GTAGCC 20.05 CCTGGA 20 CTTAGA 20 AACGCT 19.94CGCTAC 19.9 CTGTAG 19.87 CACTCA 19.87 CTTCTA 19.83 TCCTTC 19.8 CAAGTA19.73 ATCAGG 19.71 TATTGG 19.66 AGTTCC 19.66 ACACTC 19.6 AATTTA 19.59ACATTG 19.58 GAAATC 19.45 TGAAGT 19.45 GTACAT 19.44 CTTTAA 19.44 CATTGA19.38 GGCTTC 19.38 CACGAA 19.33 TATCCT 19.28 ATGGAG 19.27 AATAGG 19.25GTATAA 19.24 AATAAG 19.23 GGATTC 19.19 TCTATG 19.13 ACCCTT 19.09 ACTTTA19.01 CCAATC 19 TCTGTA 18.99 GCTCTA 18.93 GATCTT 18.92 GGATTA 18.85CGTATA 18.83 ACGAAC 18.75 ATTCTT 18.75 AGGTCA 18.72 TAGAAA 18.72 CGTAAT18.7 GTACAG 18.63 ATGTAA 18.6 TTCATG 18.6 AGTTTC 18.56 TAGTTG 18.52TGGACA 18.5 ATTTGC 18.49 CACCGC 18.45 CTCTAT 18.44 CAATCT 18.42 GAGAAG18.39 ACATTC 18.38 ATTTGA 18.37 TTGCAA 18.35 AAGATT 18.34 AAAGGA 18.34ATTGTA 18.33 TTAAAA 18.28 ATATCG 18.27 ATAGTG 18.25 GAGACT 18.19 GCTTAA18.18 TGATTA 18.16 GGATCC 18.16 AGCACG 18.12 AACCGC 18.1 TTGCTG 18.05CCAAGG 17.94 AGGCTT 17.91 CGCAAA 17.91 CCGATA 17.87 TCAAAT 17.85 CCGAGA17.85 GCCATT 17.84 GCCATA 17.82 GCACTA 17.75 ACTCTG 17.67 AGTAAG 17.64CGCTCA 17.58 TATCCC 17.54 AACTCT 17.47 TCCACG 17.46 GGAGAC 17.43 CTTGAA17.42 TCTCAT 17.31 TAGCGA 17.31 CTAAAG 17.28 CACTCC 17.24 CCGTAT 17.21GAGAAA 17.2 AACTTT 17.19 CACTCT 17.18 GACTCC 17.16 GCACCC 17.12 TTATCT17.12 TAGCCT 17.07 CCTACC 16.97 TAAGAT 16.95 GCAATG 16.95 GGTAAA 16.95AAAATT 16.92 AACGGC 16.9 CTATCT 16.81 TATCTC 16.81 GCTCCC 16.8 CTGACA16.79 CATGGC 16.78 GACCTC 16.77 CCTTGA 16.76 CTCATC 16.72 CACGGA 16.69CTATGT 16.65 TAGAAG 16.62 CATAAG 16.58 GGAAGC 16.58 CGCAGA 16.48 AACGCA16.48 CGAAGA 16.41 TAACCT 16.4 CTGATT 16.33 CAGGCA 16.28 GAAAAG 16.25CCCAAC 16.24 TAGTGA 16.23 TTGCAG 16.2 TGAAGG 16.18 TTTGAA 16.15 TACCTT16.14 GCACAC 16.12 ATGACC 15.97 TTAAGC 15.91 GTTGCT 15.9 CATGTA 15.9ACGACC 15.86 CAGGTT 15.84 AAAAGT 15.82 AGACCA 15.79 GCTTGA 15.71 GATGTA15.67 TGACAT 15.66 TTCTCC 15.65 TTAGAA 15.63 TTAGAT 15.61 ATTTTA 15.6TTAAAT 15.52 GGTACA 15.49 CATCGC 15.48 GCCATC 15.39 AATTTT 15.39 TCAATC15.38 ACCCAA 15.38 CTGTTT 15.36 CCAGAG 15.35 AGAAGG 15.33 TCATTT 15.32CCAGTC 15.26 AGTAGG 15.25 TGCAAG 15.23 AGGATC 15.22 GACAAC 15.19 TCCTCC15.19 TCAATT 15.18 TCAAAA 15.15 CCTGAT 15.13 ATCCGC 15.08 GACCTT 15.07TTATTC 15.07 GCTAAG 15.01 CTCAAG 14.96 CAGGCC 14.89 ATGTAC 14.83 CTTCTG14.71 AGACAT 14.69 TAAGTA 14.61 TTGAAG 14.6 ATGTTA 14.54 TGGAAC 14.52GGCTCC 14.47 ATAAGG 14.45 CTTATT 14.45 ATCCTG 14.42 TGTTTA 14.41 TGAGAA14.39 CACGCC 14.39 CCATGT 14.39 ACGCTG 14.36 TCCAGT 14.34 CTACAT 14.31AGTGCA 14.28 AATCTT 14.25 GGCTCA 14.24 CCCTAT 14.21 CCAGGC 14.21 CTGGAG14.2 ACCCGC 14.16 GGTATA 14.14 GACTTC 14.11 AAGAGA 14.08 GCTTCC 14AGCGCT 14 AGACTT 13.99 AAACGC 13.99 TCACCG 13.98 CACGCA 13.93 CCCAGG13.91 CTCTGC 13.88 CGAGAA 13.83 TATAGA 13.82 AAAGCG 13.82 GAGTAA 13.8GATTGA 13.77 TTGAAA 13.74 TAATTT 13.71 AGTTGT 13.57 GGAGTA 13.54 TAAACG13.52 CCGCTG 13.48 GGCTGC 13.46 GGTACT 13.42 GTGCAA 13.36 TCTGAA 13.23TCCAGG 13.15 CTTTAC 13.11 GGAAAA 13.07 ATCCTT 13.06 GAAGGT 13.04 GATTAA13 CAATTG 12.98 CATGCC 12.96 TCTTTA 12.95 GATTTT 12.9 TTTGAT 12.87CCACTC 12.84 TGTACA 12.83 TATGCC 12.83 GCTGCC 12.82 ATGGTT 12.82 GTTCCA12.79 ATCCCT 12.79 ACTAAG 12.76 ATTCTC 12.75 AACCTC 12.75 CCTATG 12.71GAATGA 12.69 ACAAGT 12.63 TACTGT 12.62 AGGTAA 12.62 AACGAC 12.6 TCCGCT12.59 TCAAAC 12.55 GCACTT 12.49 AATGCC 12.48 ACGCTT 12.45 CAACGC 12.44TAACTC 12.43 TCTTAC 12.42 CTTCCC 12.42 ACACTT 12.38 TTTTAA 12.23 GAACCG12.23 GGGAAT 12.21 TTCTCA 12.16 TGCTCT 12.15 GTAGAC 12.13 TTTTTT 12.1GTTTCA 12.07 CCCAAT 12.04 TTCAAG 12.03 TTGAAT 12 AGTATG 11.99 TAGTTT11.98 CGACCA 11.98 GCATGA 11.98 CAGGAC 11.97 GCCTCA 11.96 GTCTAT 11.95CTATCC 11.89 TGCCAT 11.88 CGATCA 11.82 AAGGAT 11.76 GTGGAT 11.71 CCATGG11.69 TCAACC 11.69 TCCCAA 11.68 GCTGAC 11.66 TCAAAG 11.63 GACACT 11.61TCCAAG 11.61 CGGCTA 11.53 GCCATG 11.51 GCCCAT 11.46 GAGCCA 11.41 GAAAGA11.4 GCGTAT 11.39 AAACGG 11.38 CCCAGT 11.36 ACACGT 11.35 TTCCCC 11.35GGCACC 11.33 AGCCGG 11.32 TTAAAG 11.31 CTATAG 11.27 ATCTTG 11.27 TACTGG11.23 CTCAAT 11.2 GCTAAA 11.18 GGTTAT 11.16 TGCCAC 11.14 GAGACC 11.07GTTACC 11.04 AGGAGT 11.04 CCGCAG 11.03 CAAATT 11.02 CTTCTC 10.99 TATGTT10.99 AATTTC 10.99 ACCGCT 10.99 CCCGCT 10.9 CGATTA 10.87 ACATCG 10.86CCGGCT 10.85 TAGATC 10.82 AAGTTG 10.82 CTTGAT 10.79 TACCGC 10.78 AAAGGC10.74 GATCTA 10.72 TCCCCT 10.64 GATAGT 10.62 GGATAA 10.61 TGAGTA 10.57GGAGTT 10.54 ACGCAC 10.52 CCCATT 10.51 TGTAAC 10.49 GATTTC 10.48 TAACCC10.46 AATGTA 10.46 ACGGCC 10.46 TGCAGG 10.44 CTGTAC 10.44 AACATG 10.43ACTGGT 10.38 AAGGCC 10.36 TAAAGG 10.29 TATTGT 10.25 GGAGGA 10.19 AAGTGA10.18 ATTTGG 10.18 TGTTTT 10.17 CAAAGT 10.16 AGTCAC 10.14 CTGAGG 10.12CTAGAT 10.11 AATTGG 10.08 GGAAGA 10.08 CTCTTA 10.04 CTCTCA 9.99 GAACTT9.97 AGAGAA 9.94 GAGGAT 9.93 GGGAAA 9.93 CCCTGA 9.92 CCAATG 9.9 TCATGA9.89 CCTTCT 9.88 TCATTG 9.81 TACCCC 9.78 TTCTGA 9.75 AGAACG 9.72 ACGCTA9.69 CTCCTA 9.69 TCCGCA 9.59 TTCACG 9.57 CGAATA 9.54 ATTTTG 9.43 GCCACA9.39 CCTAGA 9.37 TTATCC 9.33 AGGCAC 9.29 GGCAAA 9.28 AACCCG 9.28 GTTAAT9.27 AATGGC 9.23 GTATAC 9.16 CAGTCT 9.12 CTCAGT 9.12 TTTATG 9.09 TGAGCC9.05 GGTGAA 9.04 TAAAAT 9.04 CACACG 9.02 GTACTT 9.02 TTACCG 9 GCCAGA8.99 TCGCTA 8.97 GGCTCT 8.95 GACAGA 8.93 GGAATT 8.9 TATTCT 8.89 CCGCAC8.89 TGCCAA 8.87 GCCAAC 8.84 GATCCT 8.82 ACGCCA 8.74 AAAAAG 8.73 CCAAAC8.69 TAACCG 8.68 TTGAGC 8.68 GCATTG 8.65 CACTGG 8.65 GTAAGA 8.62 GACAAA8.62 CCCTTC 8.61 TTAATC 8.61 GTACAA 8.54 ATAAGA 8.53 AATCCG 8.5 TTCTTC8.39 CATGAG 8.38 GAAATT 8.32 CATACG 8.31 TCTGAT 8.28 GACCAA 8.27 TAAGAG8.24 GGATTG 8.2 CAAATG 8.17 CCACGG 8.17 GAGAGA 8.12 GCTTAG 8.08 CAGCGT8.07 GTGCTA 8.07 TTAACT 8.05 TGATCC 8.04 AATGAC 8.04 GTAACC 8.01 CTCAGG8 CGATAC 7.98 CTTTTC 7.89 TTCAGT 7.81 CCCCTT 7.75 TGCACG 7.71 TTCTTA7.71 TAATGC 7.7 CCTGAG 7.69 TATCCG 7.64 GACATC 7.64 GACCCC 7.61 CTTTGA7.6 TTAAGA 7.56 CACGAC 7.55 TAAATT 7.54 ATTGAC 7.51 AGAAAG 7.5 TTTGCT7.5 CCAAAG 7.46 CACGGC 7.4 GTTTTT 7.39 TGTGAA 7.37 GTAATC 7.37 CGTATC7.36 TACGAT 7.3 GGACAT 7.28 CCCTTA 7.23 GATTTG 7.22 ATTTGT 7.2 ACATGT7.19 CACGCT 7.18 TGCTGG 7.14 CACCGA 7.05 ATCCGA 7.01 TAGTTC 6.93 CTGGAC6.9 CCTCAC 6.9 TGAATG 6.89 GCCCAG 6.83 CGGCTG 6.82 CTTGTA 6.77 AATCTC6.73 AAGAAG 6.68 GAATTG 6.67 AAGGAG 6.63 TAGGCT 6.62 TTTGTA 6.58 TTCTAA6.55 TCTCAG 6.51 ACCCTA 6.51 TTATGC 6.47 CTGGGA 6.46 TTTGGA 6.43 CTTTGC6.39 GGAAAC 6.38 AACCGA 6.33 ACGATG 6.33 GCTACG 6.32 CTTTAG 6.28 GCAGGC6.25 CTGCCC 6.22 TTCTTT 6.2 GCACTG 6.19 ATAGTC 6.11 GCTCAC 6.11 ATTGGT6.09 GTACTG 6.09 GGTATC 6.07 CCCAAA 6.05 CATTGT 5.96 GTGCAC 5.86 GTTTTA5.81 GCAAAC 5.79 CGCACC 5.79 CTACCG 5.78 GGGATA 5.77 ACAGGT 5.76 GCTGAG5.75 AAATGT 5.7 TGTAGT 5.67 TGATGG 5.64 ATGCCC 5.63 TTTCCC 5.63 GCCAAT5.59 AAGGTA 5.58 GTATCC 5.56 TGGACC 5.48 AGGCAT 5.46 GATGGT 5.44 TTCCTT5.44 TGGAAG 5.39 CCTATC 5.33 CGGACA 5.31 AGGGCT 5.22 TTTAAC 5.22 TTGTAA5.21 ATAGGC 5.18 TGTTAA 5.15 TGACTA 5.12 CCCCTA 5.11 AGATGT 5.1 GACAAT5.09 GATCAA 5.07 GCCAGC 5.05 TCATCC 5.04 AGTTAA 4.96 TCTCAC 4.95 ACGGCT4.94 TCTATA 4.87 GTAGGA 4.85 TTTCTA 4.85 CAGAGG 4.84 TTTTTG 4.77 TCCTAT4.76 GAAGGC 4.74 TCAGAC 4.73 GCAGCG 4.71 AGTGGA 4.7 CCACGC 4.69 TTGTTA4.62 CTTAAA 4.62 ACTGCG 4.61 GTTCAC 4.59 TCAAGG 4.58 AGGATG 4.56 CCCTGT4.46 CAAAGG 4.45 TTTAAA 4.39 TTATGG 4.38 CTAGAA 4.37 CCGTAA 4.36 TAGCCG4.36 ACTTTG 4.36 GACTGA 4.33 TCACAC 4.31 GGTAGA 4.27 GACTGC 4.25 AGATTG4.24 CGGCTT 4.23 ATGTCA 4.23 TCTTGA 4.2 CTTTTG 4.2 TGTAAA 4.2 GCTTTG4.19 CCAAGT 4.16 TGTACC 4.15 AAAGTT 4.14 ACCGTA 4.1 TACGAA 4.04 CTTATC3.94 CCTCAA 3.94 ACCCGA 3.93 GTTGAT 3.93 TGCTGT 3.92 GTTCAG 3.91 TGGTTA3.91 AAAACG 3.88 GCGCAG 3.86 CCTTTC 3.85 TCTCAA 3.85 ATCTAG 3.83 GAGATT3.8 ACGACA 3.75 TAGACT 3.73 TGTATG 3.7 GCTAGT 3.7 TAAGCC 3.7 AAAGGT 3.68CTAAAT 3.65 CAGTGT 3.61 GAGTTC 3.56 AGGGCA 3.54 CGCTTC 3.53 TACCGA 3.51TCCTCA 3.51 AGCAAG 3.5 GAAGCG 3.49 GCCTTA 3.43 TTAGTT 3.4 ACCGAC 3.39GCAGGA 3.39 ATGCGA 3.38 ACGAGC 3.35 GCAGGT 3.33 AGGGAT 3.33 CAGGGC 3.29AAGGGA 3.26 AGCGGC 3.25 GACCCT 3.25 CGCCAT 3.18 GTGAAA 3.17 AGAGGA 3.16GGGATT 3.16 ACGGAT 3.13 TGCTAG 3.1 TATGCG 3.06 GACCTG 3 TTGGAT 2.99TACTTG 2.98 GACAAG 2.95 TATGAG 2.93 GACTCT 2.87 GTTGTA 2.85 GTCACC 2.84CATGTC 2.82 TGGTAC 2.78 CTCCTT 2.78 ATCTGT 2.78 AGGACT 2.76 GGTAAC 2.76TCCCAT 2.75 CAATTT 2.73 GCTGGT 2.69 ACGATT 2.63 CGAACT 2.6 GACACG 2.58ATGTGA 2.58 CCTAAA 2.57 TGGCAT 2.49 CTGGTA 2.48 ACTTTC 2.47 GAGTAG 2.46TTTCCT 2.4 CCACAC 2.39 TGTTCA 2.38 AACTTA 2.38 TGTTGA 2.35 GAAAGG 2.33ACGGCA 2.33 GAGCCG 2.32 TCTTAG 2.32 CAATGT 2.29 GTCCAT 2.28 ACCGCA 2.24CTCCTG 2.22 CTAGAG 2.19 TCATTC 2.19 AAGGCA 2.18 CCCTTT 2.15 AGGTTC 2.11CTTAAC 2.1 TTGACC 2.07 GCTTTC 2.06 AGACAA 2.06 TTTCTG 2.02 GGTGAT 2.01CCTCAT 1.99 GAGAGC 1.95 GCCTTC 1.91 TGATGC 1.88 AGAGGC 1.87 GATGAC 1.87GTTTCT 1.83 TAACGA 1.8 CTTACC 1.79 ACTGAC 1.72 ACGCAA 1.7 CGAATC 1.69GGACAG 1.64 GCCGAT 1.64 TGGGAA 1.62 AGACGC 1.6 TTACCC 1.58 CAACCG 1.55CCCTCC 1.51 TTCAGG 1.48 TCACGA 1.48 TGCTTT 1.44 AGGGGA 1.42 ACGGAC 1.41CTCCCC 1.38 ACCTTG 1.35 AGAGTA 1.3 GCCAAA 1.29 AAAGTG 1.28 CCCCTG 1.21TTGAAC 1.21 GATGAG 1.21 GCGCTG 1.2 TCAATG 1.17 CTTGGA 1.16 AGGGAA 1.14GTTGAA 1.14 AGAGTT 1.08 AGACGG 1.08 TTGGAA 1.05 TCTCCC 1.02 CTCTAA 1.01TCTGAG 1 TCGATT 0.95 ACGAAT 0.83 TGGAGG 0.82 CATGGT 0.82 GAAGAG 0.81TTCCTG 0.78 CGCTTT 0.75 CGGAGA 0.75 GATAAG 0.72 GGCATT 0.71 GGCAGT 0.67ATTCGA 0.67 CATTTG 0.59 TCTTAA 0.58 ATTGAG 0.55 TTTTCC 0.54 CAAAAC 0.47AGTGAC 0.47 GCCTCC 0.45 GACGCT 0.39 CATCCG 0.39 CTATGG 0.38 TCATGG 0.37GGGACA 0.36 CCTGCC 0.36 CAGGGA 0.34 TTCGCA 0.32 AAGGTG 0.25 GATGTT 0.2TTTTAG 0.18 TGGTGA 0.16 CTGTGA 0.14 GGCAGA 0.11 GTGTAT 0.1 CCCTAA 0.09TCTCCT 0.06 ACTCGA 0.05 TACCTC 0 AATCGC −0.05 ACTTAA −0.05 CTCAAA −0.06GCCCCC −0.1 GGTTAA −0.11 GCGAAA −0.15 CTAGTT −0.16 TCCCCC −0.21 AACTTG−0.22 CTCCGC −0.27 AAACGA −0.29 TGCCCC −0.34 CGCTGC −0.35 AAAAGG −0.35TGCATG −0.38 CAGACG −0.39 TGACAC −0.39 CGATGA −0.4 TTAAGG −0.41 TTGGAG−0.41 GCCCAA −0.41 AGGTTA −0.42 ATTTAG −0.45 AGATTA −0.46 AGGTTT −0.49GCCTAT −0.53 TCATGC −0.55 CTCATG −0.58 CAGTCC −0.59 GTATGA −0.64 CCTCTA−0.65 CATTCT −0.65 CCGACA −0.73 AGTTAG −0.81 GCCAAG −0.86 ATTCTG −0.86GAGTTG −0.88 AAAGAG −0.91 TGTGCT −0.96 TCTAAG −0.96 AAACTT −1.01 GCGGCT−1.04 TTAGAC −1.04 TTAAAC −1.08 AAGGTT −1.14 AGTTGG −1.15 AGAGGT −1.2CCCTAG −1.2 CCGCTC −1.21 GCATGG −1.24 GCTAGA −1.26 ACGATC −1.27 CGTGCA−1.27 TTTAGC −1.32 CTCATT −1.33 CGCAGT −1.35 AATTGT −1.36 TGACAG −1.37ATGCCT −1.38 AAGTTC −1.41 CTTGAC −1.45 TTTTGA −1.48 ATAACG −1.48 GCATTC−1.49 ATCGGC −1.51 GTAATG −1.54 TAAACT −1.55 GAATGG −1.56 AATTTG −1.6CCTCCC −1.61 CGGATT −1.62 TTGAGA −1.64 GTGAAG −1.66 GCCCCT −1.7 CGTTTA−1.73 GAGGAA −1.76 CGTTCA −1.77 TTCGAA −1.81 ATCGAC −1.83 TTTTTC −1.87TGCGCA −1.89 ACCGAA −1.9 CTGCGC −1.93 AAGTCA −1.93 TTACGA −2 TGGACT−2.05 TACGCT −2.06 GAGGCA −2.1 TTGATG −2.12 ACCGAT −2.13 TACTCT −2.17CGCCAG −2.18 GAGTTA −2.18 CACGTT −2.2 CTGCGA −2.22 GTGCTT −2.23 AATGAG−2.24 AGTGTA −2.25 CTTATG −2.26 TCTCTG −2.27 CCTAAT −2.29 GGAATG −2.29CCATTG −2.34 CGATAA −2.35 ACTTGA −2.35 TTGGTA −2.35 TAGAGA −2.36 GACATT−2.38 GGGAAC −2.38 TGACAA −2.38 GTGCAG −2.42 CGGCTC −2.43 ATTGTT −2.45ATGAGT −2.46 GGATGA −2.48 GTTCTA −2.49 GTTAAA −2.5 ATGTTC −2.57 CCTAGC−2.61 CCCTAC −2.61 AGAATG −2.65 CGAAGC −2.7 CGGTAA −2.71 CTAATC −2.72ACCTAA −2.76 GCGCCA −2.8 GTCCCA −2.83 CGAGCA −2.88 TCAGGT −2.9 AGAGTC−2.92 GAGGAC −2.92 ACGAAA −2.95 AGGCAG −2.97 GGACCT −2.98 TCACTC −3.01GACTGG −3.03 CTTGAG −3.03 CGAGCC −3.07 GGCTGT −3.1 GCCGCT −3.11 GGACAA−3.11 TACCCT −3.12 GTCAGC −3.12 CTGTTC −3.18 CCGAAT −3.21 AGAGCG −3.21ATGGTG −3.29 TCCTTT −3.3 CATGAC −3.31 TAGACC −3.31 GGACTC −3.32 CCCTGC−3.32 GGAAGG −3.35 GGTTCT −3.38 GCAATC −3.41 AGTCTT −3.46 TACGGA −3.49CGCACT −3.51 GCCTGC −3.57 GGACCC −3.57 GCCTTT −3.58 TTTAGT −3.6 GGTGCT−3.6 CGACTC −3.65 GGATAG −3.69 GGATGC −3.7 ACTCTT −3.73 ATTGCC −3.84TGAACG −3.84 CTTTTT −3.89 GAATGC −3.91 TATAGG −3.92 GTATAG −3.93 GAGCGC−3.96 ATTGTG −3.97 TCAGAG −3.97 GGGATC −3.98 CCGCCA −4 TGTCCA −4.01TGTTCT −4.03 AGGCCA −4.04 CCTTAC −4.05 TTTTCT −4.07 CATCGA −4.09 AGCGAA−4.12 AAAGGG −4.12 GGGAGA −4.13 CTGAGT −4.13 GAAGTC −4.15 CGTAGC −4.16CGGCAC −4.18 TGCGAA −4.19 TCTTTT −4.21 ACGGAA −4.22 CCGACT −4.25 ACCTGT−4.26 ATCGTA −4.29 TATGGT −4.29 TAATCG −4.31 CGATTC −4.32 GGGAGC −4.38CTCTAC −4.38 CGTACA −4.41 CAAGTT −4.42 TAAGGC −4.46 AAGCGA −4.46 GGTACC−4.48 GACAGT −4.49 CCGCAA −4.53 GCTAAC −4.61 TCCCTA −4.62 CAGGTG −4.63CAATGG −4.64 TAGTCA −4.67 TAGACG −4.67 CGTGAA −4.7 AGACGA −4.7 AAGCGT−4.74 TGGGAT −4.81 CCGAAG −4.83 CGAAAA −4.87 AGCCCG −4.93 GGCTGG −4.96GTCACT −4.99 CAACGA −4.99 TGACCC −5 GCCGCA −5.04 GTTCAA −5.06 TCGCTG−5.07 GTGAAC −5.11 CCTTAG −5.16 ATAGGG −5.17 CAGTGC −5.18 AGGCGA −5.2CGAACC −5.2 ACTCCG −5.21 CTCCTC −5.24 GGTCCA −5.25 AAATTG −5.27 CAAGTC−5.27 TACCCG −5.28 CTTTCC −5.29 GCACTC −5.29 TTGGCA −5.3 ACTTGC −5.32AGTCCC −5.32 TGGCAC −5.33 GTGGAA −5.33 GGCCAT −5.36 GCGGAT −5.39 GCGCAT−5.4 GGGGAA −5.4 TCTAGA −5.4 ACTTGG −5.44 TGCGAT −5.45 GCGATA −5.45TGCCCA −5.45 TGGCTT −5.48 AGAGAG −5.48 TTGCTT −5.51 AATGTG −5.57 TTACGG−5.57 AAGGTC −5.59 TGAGAC −5.62 GACTGT −5.69 TTAGTG −5.71 CATTGG −5.71CAGGTC −5.73 TCCCTT −5.8 CGAATT −5.82 AATGTT −5.82 GGCAAT −5.83 TAGGAC−5.91 TACGGC −5.94 TCTTCT −5.95 GGGCTC −5.97 TCGCAT −5.98 CTAGGC −5.98CCTTTT −5.99 CCAGTG −6 CACGAG −6.01 TCCTAA −6.03 TAGGCA −6.08 TCTAAC−6.1 CACCCG −6.13 CTACGG −6.14 AGGTGC −6.16 CCCATG −6.17 ACGCCC −6.17CGATCC −6.18 GAAACG −6.2 ATGTGC −6.21 GCAAGC −6.24 AAATCG −6.25 CCTCTC−6.29 ACCGGC −6.31 TTTAGA −6.33 CGACTG −6.33 AGGCAA −6.33 GGACAC −6.35TAGCCC −6.37 TCTGGA −6.37 TAAAGT −6.37 TGAGTT −6.37 AAACCG −6.45 ACCCGG−6.51 CCTGAC −6.51 AAATTT −6.52 AACTCG −6.52 AAGGGC −6.52 TTTTGC −6.54GGAAGT −6.61 GGTTAC −6.66 TCGTAT −6.68 GTTCTC −6.7 GGAAAG −6.72 TCCTTG−6.72 GCGAAT −6.75 AGTCTC −6.77 GGCACT −6.8 GCTCTG −6.8 CTACCT −6.8TTGACA −6.81 AGCGTA −6.81 AGCGTT −6.83 TCGCAG −6.85 CGAAAT −6.88 GCCCTT−6.9 CATCGT −6.91 AATTAG −7 GACGAT −7 AACCGG −7.03 TTGCCA −7.04 CTAGCC−7.1 CACGGT −7.11 CTCTTC −7.13 AACGCC −7.15 GTTATG −7.15 ACTGTG −7.15TAATGT −7.16 CCAACG −7.21 GCCTTG −7.21 CCTTGT −7.23 TTCTGC −7.23 TAAGAC−7.23 GCTGTG −7.24 CCCCTC −7.25 GACTAA −7.25 CGCTCC −7.27 GCGACT −7.27TTCCCT −7.28 CGCCCA −7.29 TGCGCT −7.33 CCCCCC −7.34 TTAGAG −7.34 CCTGTT−7.36 TCTTCC −7.38 CCATCG −7.38 TCTAAA −7.39 CTAATT −7.42 AAGCGC −7.42CTCTGT −7.48 AGGCCT −7.48 TAGGAA −7.51 GTTCTT −7.54 GTATTC −7.63 ACGAGA−7.68 ATGTTT −7.68 GGGTAT −7.76 ACTAGG −7.77 CGGAAA −7.79 ATGGCC −7.82GTTATC −7.85 TCGACT −7.87 CTTCTT −7.9 AACGAT −7.91 GATCGA −7.94 CTCTAG−8 CTAACC −8.08 CTAGGA −8.08 GATTGT −8.1 CCCGCA −8.1 CGAGTA −8.11 TGGGCT−8.15 GGCTTA −8.19 TCGGCT −8.24 GATGGC −8.25 ACGCAT −8.3 CCGCTT −8.3TGGCTG −8.32 ACTCTC −8.35 GCCCAC −8.37 CGCTGG −8.37 TTGCTC −8.38 TGGTAG−8.38 CTCTGA −8.49 TACTCG −8.59 TGAGAG −8.6 GCACCG −8.61 ATGGGA −8.69TGACTG −8.7 CGATTT −8.72 CGGAGC −8.74 CGGATC −8.78 AGTCAA −8.79 TTCCTA−8.79 CCTAGG −8.79 GTTGGA −8.82 AGTCTG −8.83 CAAGGT −8.85 AATTCG −8.91ATTCGC −8.93 GAAAGT −8.99 CTAGAC −9 AACGAA −9.02 CGACAA −9.03 GCCTAG−9.04 AAGTGC −9.06 GGTGTA −9.06 GATAGG −9.08 TTTGCC −9.1 TTTAAG −9.1CCCCCT −9.1 CGATAG −9.13 ATCCCG −9.15 GTCACA −9.21 GTCCAG −9.24 CAAACG−9.25 AGGCCC −9.29 AGGGAC −9.3 CTGACC −9.3 GCTGCG −9.34 TTTCTC −9.36CGACAG −9.37 TGAGGA −9.41 CCAGGG −9.43 AGTCTA −9.43 GCCGAA −9.48 TCCCTC−9.49 AAGTCT −9.51 AGGGCC −9.52 GCAGTC −9.54 ATGTTG −9.55 GTAAAC −9.56GAGTTT −9.58 ATGCGC −9.6 CTCCCT −9.65 TTTTGG −9.67 GTCAAT −9.69 TAGGAG−9.7 CTTCGA −9.71 AGTTTA −9.73 GTAAAG −9.78 CGCCAC −9.81 GACAGG −9.82AGGAAG −9.83 ACGTAT −9.85 GAACGC −9.88 AAGAGT −9.91 CACTTG −9.92 GCGATT−9.92 CGCCAA −9.93 GCTTAC −9.94 TGACTT −9.94 CATGTT −9.95 TGATTG −9.97TCACGG −9.98 TCGAAT −9.98 CTCTTG −10.02 GTGATT −10.03 GAACGA −10.03TGTTCC −10.05 TGTTTC −10.07 TCTTAT −10.08 GAGACG −10.09 CGGTTA −10.12GCATGT −10.13 GGATGT −10.15 CCTTGG −10.18 GAATCG −10.2 GGGCTG −10.21TAGAGT −10.25 TAGCGG −10.25 GCAGTG −10.25 GTCCAC −10.28 GAGTAC −10.33CCACCG −10.36 CGACAT −10.37 GGGGAT −10.38 CGCTAA −10.4 CCGTTT −10.41TCTAGC −10.42 GGGATG −10.45 CTGTGC −10.48 CTAAGG −10.48 TTGATC −10.5ATTGGC −10.52 AGCCGT −10.56 ACTGGG −10.56 CTGGCT −10.58 ACGCCT −10.59ATACGT −10.63 GGTAGC −10.65 TGTCAT −10.65 GATGCC −10.66 GGTTTA −10.7GTGCTC −10.84 TAAGGA −10.86 CTTAAT −10.91 GATCCG −10.94 CGAGAT −11GGCGAA −11.02 CCGCAT −11.03 GGCGCT −11.04 GCACGA −11.04 TGCCGA −11.07GGCATC −11.1 TCGGCA −11.1 GATTAG −11.14 TCCTTA −11.15 CTAAAC −11.17CGGAAG −11.23 CTTTGT −11.26 TTAGGA −11.27 CCGGAT −11.36 ATTAAG −11.38GTGCTG −11.41 CTCTCC −11.45 TATTCG −11.47 GCCCTG −11.51 TCGCCA −11.54TGTAGA −11.62 CTAGTG −11.62 CCGCCC −11.66 CAAGCG −11.66 GGTGGA −11.74ATTAGG −11.75 GCCTAC −11.77 CTCACT −11.78 AAGCGG −11.79 AACCGT −11.81AGATCG −11.85 TGACTC −11.92 TTCTTG −11.94 ATCGCC −11.99 ATCGAA −11.99GGTTTT −12.02 TGGCAA −12.04 CGCCTT −12.06 TTGTAG −12.07 ACTTGT −12.08TGGTTT −12.08 ACTCGG −12.09 TATGGC −12.1 TTGGTT −12.12 GCGATG −12.19CAGGGT −12.2 AGTTTG −12.24 TAATCT −12.24 AAACGT −12.25 GGCAAT −12.28CCCTCT −12.28 GGGCAT −12.33 AGTGGC −12.33 GCCAGG −12.34 TAAGGT −12.35GGCCTT −12.37 GGGAAG −12.37 TGCCTA −12.4 CCGTCA −12.43 GTATTG −12.44GTGACA −12.48 CGGCAG −12.51 TGTGAT −12.53 GACGCA −12.56 CAAGGG −12.58GAGTCA −12.63 GCCGAG −12.66 CTTTCT −12.68 GACTTT −12.69 GGTCAA −12.72TCGCAC −12.75 TCTTGC −12.82 CCTTTG −12.82 TTCGCC −12.88 TGGTCA −12.91GCGCTC −12.95 GAAGTG −12.95 GCCTCT −12.96 AGGTGG −12.96 CAGTGG −13GTACCC −13.02 TTCCTC −13.04 TCGACA −13.05 TGGCAG −13.07 CCGAAA −13.09CTGCCT −13.11 ATGGGC −13.12 ACCGAG −13.13 CGTAGA −13.16 GGGCTT −13.18CCGAGC −13.19 GACTTG −13.23 CTGACT −13.26 GAGGGA −13.28 AGTCGA −13.32CCCGAG −13.32 CTTCCT −13.32 TCACGC −13.37 TAGGTG −13.39 CCTCTG −13.41GCGACA −13.46 GCTAGC −13.46 TCATCG −13.48 CCCGCC −13.49 GTCCAA −13.5TGGAGT −13.55 ACGAGT −13.6 CCCGGC −13.6 ACGGTA −13.65 TCCTGG −13.65CGATCT −13.73 CAATCG −13.76 CTACGC −13.79 ATCACG −13.84 CGCTCT −13.89CCCGAT −13.92 CGGTAT −13.94 AAGTCC −13.95 GGCATG −14 ATGAGG −14.05AGGTCT −14.05 CTTAGT −14.06 ACTCGC −14.08 ACGAAG −14.09 GGGACT −14.1AAGACG −14.11 TCCTGC −14.11 GCTCTC −14.12 TCTACG −14.14 TTGAGT −14.15TCGAAC −14.16 CCCTTG −14.27 GTTCCT −14.28 GTCTCC −14.29 ACCGTC −14.35TCTTTG −14.39 GGTTCC −14.39 GTTCCC −14.42 CGCTGT −14.51 CAACGT −14.53CAGGCG −14.56 TACGCC −14.59 CGAAAC −14.6 TCTTTC −14.65 TGCCCT −14.67GCCCTA −14.68 GTTTTC −14.68 GTATGC −14.7 GAAGGG −14.72 CGAAAG −14.79GATTCG −14.79 CGATGC −14.9 TGAGCG −14.92 ACGCGG −14.93 CTCGAG −14.94TGCGGA −14.95 ATGTCC −15.01 CGGCCA −15.02 ACCTAG −15.05 GTCAAA −15.06GTGCCA −15.08 CCCCGA −15.11 CTGGCA −15.12 AAGGCG −15.13 GATTGC −15.14TTTGAC −15.14 GTAGGT −15.16 GTTGTT −15.17 CCTAAC −15.17 GGACTT −15.18CGTAAA −15.2 TCATGT −15.21 GGGACC −15.22 GGGCAG −15.23 CTGGTG −15.25GGATGG −15.27 CCGTTA −15.31 GACGCC −15.32 CGCATC −15.33 ACGCTC −15.33AAAGTC −15.35 GGGGCA −15.38 CTCGCA −15.38 GCACGG −15.39 AGCGAG −15.4ACTGGC −15.44 CTGTCA −15.51 AGCGTC −15.52 GAGGAG −15.53 GTGTAA −15.58TTGTAC −15.6 TCAGTG −15.65 GGCGCA −15.71 GCGAAC −15.71 TCTCTA −15.73CCCGAA −15.75 TGAGGC −15.76 CCCCGG −15.78 CCTCGA −15.83 TATCGG −15.85ATCCGT −15.86 AGCGGG −15.87 CCCACG −15.87 ACTGTC −15.88 GTTTAA −15.92TAGTGG −15.97 AATGGG −15.99 ATCGAG −15.99 GTCCTT −16.01 AACGTG −16.03CGCAAG −16.03 GGCCCT −16.05 CACGTA −16.06 TAGGGA −16.09 CGGCAA −16.12CCTAAG −16.15 TCGAGA −16.16 GCCTGA −16.16 GACCCG −16.19 GTTAGA −16.27TGCTTG −16.27 TCGAGC −16.29 ACGGTG −16.32 TCGATC −16.34 CAGGGG −16.36GAATGT −16.41 TTGACT −16.46 TCAGTC −16.47 GCTCGA −16.48 AATCGT −16.48GCCCTC −16.49 GACGGA −16.49 AAGAGG −16.52 CGTTAC −16.52 ATCCGG −16.55TTATGT −16.55 CTTCGC −16.56 GAGTCC −16.57 GAGAGG −16.6 TGTCTT −16.61AGAGTG −16.64 ACCCCG −16.65 TAACGG −16.65 CTCGGC −16.66 TAGAGG −16.67CTTCCG −16.75 AACGGA −16.76 AAGTTT −16.76 GCCTGT −16.77 AGTCCT −16.79GAACGG −16.8 GGCAAC −16.84 CTCGGA −16.85 TCGATA −16.85 ATGGGG −16.85GGAGGC −16.88 CCGCCT −16.93 CCTCCT −16.95 AAGGGG −16.95 ACCGTG −17GCCTAA −17.04 TGGGAC −17.08 TGGATG −17.08 TATCGC −17.09 GGACTA −17.1CGAAGG −17.11 TCTAGT −17.14 GTCAAC −17.15 TTCTAG −17.16 CGAGAC −17.19AAGGGT −17.2 GCGAAG −17.21 GCAAGT −17.22 CGGCCC −17.26 ATTTCG −17.3GTGGTA −17.33 TGGTTC −17.37 GCATCG −17.37 GTACTC −17.39 ACGTAA −17.4CTTGTT −17.4 GGACTG −17.41 GCCGTA −17.43 CCTGGC −17.44 AACGAG −17.52CGCAGG −17.55 TCTTGG −17.58 AGACCG −17.62 TGCGAC −17.65 CAGTCG −17.66GCCGTT −17.66 TAACGC −17.67 CGACAC −17.69 CCCGGA −17.72 GTCATT −17.72ATCGGA −17.74 CCGAGT −17.76 GGTTTC −17.8 CGCATT −17.82 CCTTGC −17.83TCTGCC −17.83 GCAAGG −17.84 CCCTGG −17.85 GTTTAC −17.87 AGGTCC −17.91GGTTGT −17.93 GCGATC −17.95 TCGAAA −17.95 CTTGCC −17.99 TCCGAC −18TATCGA −18 GATTGG −18.08 CGTTAT −18.09 TATCGT −18.16 TTTCGC −18.18AAGTGG −18.22 GGCCCC −18.22 GGCCCA −18.24 ACCGGA −18.25 TCAGGC −18.26CGTCTA −18.28 GTCATC −18.3 GACCTA −18.31 TTGTTC −18.38 TCCTAG −18.4ACCCGT −18.48 ATCGTG −18.49 TTGGAC −18.49 CGGAAT −18.51 CAACGG −18.61ACCGTT −18.62 CCGTAG −18.63 TGCCAG −18.65 TGTTAG −18.77 CGACCC −18.77TTGTTT −18.77 TCGCAA −18.77 ATGGTC −18.81 CGTGGA −18.81 TCCTCT −18.83TCGCCT −18.84 TCGGAT −18.89 GCGACC −18.9 ACGTTT −18.91 TTAGCC −18.92CTCTTT −18.92 ACTTCG −18.95 CTATCG −18.96 GCGCAC −18.96 TCGAAG −18.97TTATCG −19.01 TAAGTG −19.03 TGATCG −19.03 CTCGAT −19.04 CTCGAA −19.13CTCACG −19.18 GGCTTG −19.19 CGCCGA −19.19 CTTGCT −19.24 GTAGTC −19.28CACCGG −19.31 TTTGTT −19.31 TCTGTT −19.32 TTTACG −19.34 GTCCCC −19.35CGAGGA −19.35 CGGATG −19.35 CCGATG −19.37 CATCGG −19.38 GGTAGT −19.38CCGTGA −19.41 TCCGCC −19.41 TCTCTT −19.42 GGAGAG −19.43 CATTCG −19.47CGAATG −19.54 TCTCTC −19.56 GGCCAA −19.57 GCTGTC −19.65 ACCGCG −19.66GTGAGA −19.68 GGCCAC −19.7 CCTAGT −19.71 TCTTCG −19.73 GTGATC −19.73ATGTAG −19.77 GTGACT −19.79 GACGGC −19.8 AGGGGC −19.83 ATTCCG −19.84GTTTCC −19.85 GGCAAG −19.96 CGGCAT −19.96 TCCCGC −19.96 AGTGTT −19.97GCCGAC −19.99 CCGATT −20.01 ATTCGG −20.03 TACCGT −20.03 TCAGGG −20.08GTTTGA −20.11 GCTCCG −20.13 CCTGGT −20.17 CCTCTT −20.18 ACGTGA −20.22GTCTAA −20.25 TAAGGG −20.27 TCCCCG −20.29 CACGTC −20.32 GGCAGG −20.33CGTAAC −20.35 GAGGCC −20.36 TAGTCT −20.36 AGGGAG −20.39 ACTCGT −20.39CGCTTA −20.4 GCGGAA −20.46 GGCTAA −20.5 CCTTCG −20.52 TAAGTT −20.52TTGGCT −20.53 CCGGAG −20.53 ACGCCG −20.58 GTCTCT −20.59 CCGAAC −20.66AGGGTT −20.69 GGTCAC −20.72 AGTGGT −20.73 TGTCAC −20.75 CCCCCG −20.77TTCGAT −20.79 CGTAGT −20.82 GCGGCA −20.82 TCCGAG −20.86 TCAAGT −20.87CCGTCT −20.88 GGAGGT −20.93 CTGACG −20.94 TGCCTC −20.94 AGTCAG −20.95TTCTCT −20.97 CGGTTC −20.97 TGTCTA −21 TCTCCG −21.04 CACTCG −21.05TGACGA −21.15 GTCTCA −21.17 GTCAAG −21.27 CTTGGC −21.28 ACGTCC −21.28CGGTGA −21.32 TTGGGA −21.4 TCGTAA −21.4 CGGAAC −21.42 GGTATG −21.43ACCGGT −21.5 CCGGAA −21.51 TCGTTA −21.53 AATGTC −21.55 CATGTG −21.55GCGAGA −21.58 TTTAGG −21.6 GAGGTA −21.69 CCGGCC −21.71 TGTGGA −21.72CTCTCT −21.73 GTGGCT −21.74 GCCGCC −21.76 GACCGA −21.76 GGTCAT −21.8TCCCTG −21.84 GCGCTA −21.87 TCCGTA −21.87 TTGTTG −21.9 GTCCTA −21.93GCCACG −21.95 TGCGTA −21.97 TCCGAA −21.99 GCCGGA −22.01 GAGCGG −22.07TTCTCG −22.07 GACGAA −22.08 CTGCCG −22.11 CTGGTT −22.11 AGGCCG −22.12GAGTCT −22.25 ATGGCG −22.26 GGGCAC −22.28 AGTCGC −22.31 GCGGAG −22.37TCTCGA −22.4 GACCGC −22.5 CTCGAC −22.51 ACGGGC −22.53 GCGCAA −22.56CTCCCG −22.58 GTATGT −22.6 GGGCAA −22.61 ATCTCG −22.63 AGTGCC −22.66GTCTTC −22.66 CGGGAA −22.68 CGATGT −22.69 GACTTA −22.7 CGCGCA −22.71GACGAC −22.71 GGGGCT −22.72 TCCCGA −22.78 TCAACG −22.81 CGTCAA −22.81GATGGG −22.81 TGCCTT −22.82 TACGGT −22.84 TTTGCG −22.87 CGCCCC −22.92GAGGTC −22.93 ATTGGG −22.97 CGGACT −22.99 AACGTA −23.02 ACGTTC −23.04GACTCG −23.1 CTGTTG −23.16 GCTGGG −23.19 CGTTTT −23.21 TACGAG −23.26GCCAGT −23.28 TTCGTA −23.29 CCTCCG −23.29 TTCTGG −23.3 GGGGAC −23.32GATCGC −23.32 CCCGAC −23.33 CGGGAT −23.34 GTGTTA −23.34 GTAGAC −23.35GCAGGG −23.38 AGAGGG −23.41 ACGGTT −23.42 CGCCTA −23.43 GGGCTA −23.43GTACGA −23.43 TTTGAG −23.44 TACGTA −23.44 GTGACC −23.47 CTCGCT −23.49ATTGTC −23.58 TTAAGT −23.58 TTACGC −23.64 GTTAAG −23.65 CCGGCA −23.74AACGGT −23.77 CGAGTT −23.8 GGCCGA −23.81 GCGGTA −23.82 GCAACG −23.84GCGATC −23.9 CTCCGA −23.94 CGGCCT −23.97 TCCGAT −23.99 AGACGT −24.01TTTCGA −24.02 TTTTGT −24.03 ATTCGT −24.04 TCACGT −24.05 CCTGGG −24.05TGTAAG −24.09 AATGCG −24.13 CGTTCT −24.15 CCGAGG −24.17 TCTAGG −24.2TGGGTA −24.22 GTGTTT −24.23 TGATGT −24.25 TAGGTT −24.27 ACTTAG −24.29AACGTC −24.31 AGGTTG −24.34 GTAGCG −24.4 GTTAAC −24.41 TATGGG −24.43TAGCGC −24.44 CGTCAC −24.48 TTCCGA −24.5 GACTAG −24.54 TGGGGA −24.57GCGCTT −24.58 TTCTGT −24.59 GGAGTC −24.6 CGCCTG −24.62 CGATTG −24.63GGTGAC −24.68 TCGTAG −24.68 TGTCAA −24.69 GGGTTC −24.7 TTCGAC −24.76TGTGTA −24.79 GAGTGA −24.81 GACGAG −24.82 CTAGGG −24.83 GTTGAG −24.87TGACGC −24.87 CGCAAC −24.94 CGCCTC −24.96 GAGCGA −24.96 CAAGTG −25.01TGGTGC −25.01 ACGGCG −25.02 CGAGGC −25.03 TACGCG −25.05 CATGGG −25.06CTGTCC −25.07 GTAAGC −25.1 CGTGTA −25.17 ATGCCG −25.2 ACGTAC −25.24TTCCGC −25.28 GTCTTT −25.31 TCCGGA −25.33 TTGTGA −25.35 AGTTCG −25.37AGCGGT −25.47 GCCCGA −25.51 CTGGGC −25.54 TAGTGC −25.55 TTGCCC −25.57TCTTGT −25.63 TGCGCC −25.65 CGAGAG −25.69 TATGTC −25.72 TGTCCT −25.75AATCGG −25.77 TTTCCG −25.78 TATGTG −25.8 TGGGCA −25.88 GCTTGC −26.03TCGACC −26.05 TTAGCG −26.06 CCGTTC −26.08 CTAACG −26.09 GGCGAT −26.11GTTAGC −26.11 GTGGCA −26.14 CCGGGA −26.15 GCCTGG −26.17 CTTAGG −26.18AACGCG −26.19 CGCGAA −26.21 ATCGTC −26.24 CTTGGG −26.27 GCACGC −26.29GAGAGT −26.3 GCATGC −26.3 ATCGTT −26.33 GAGGTG −26.36 TTAACG −26.36CTGCGG −26.38 ACGGGA −26.47 GGCCTG −26.49 CCTGCG −26.49 AGGGTA −26.49GAACGT −26.5 TTTGGT −26.53 ACGGAG −26.54 GGAGCG −26.73 CCGCCG −26.75CCTACG −26.76 GTAACG −26.81 CCCGTA −26.81 GCTTCG −26.82 TAGTCG −26.83CGTCCA −26.91 TGAGTC −27.01 CTCTGG −27.01 ATTGCG −27.01 CGATGG −27.05GCTAGG −27.14 GGGAGT −27.16 ATGTCT −27.2 CTGGGT −27.21 GGACGA −27.23CGTTTC −27.24 ATGACG −27.27 TTCGCT −27.27 AGGGTG −27.33 CTTCGG −27.4CGAAGT −27.41 TTGCCT −27.41 GGATCG −27.41 AGGCGC −27.45 GGGTTA −27.47ACGCGC −27.48 TTGTCA −27.57 TAGTGT −27.58 GAGGTT −27.6 TGTCTC −27.6GTGATG −27.65 GGTCCT −27.65 CGGACC −27.65 TCGCTT −27.66 TCGGAA −27.69ACGTCA −27.74 TTCCCG −27.84 GCACGT −27.87 GTCGCA −27.88 CGTTAA −27.93ACCTCG −27.95 TGGGAG −27.96 CTGTGT −27.97 TAGCGT −28.06 AGGACG −28.08GGCCTC −28.1 AGTACG −28.14 TAAGCG −28.21 CTGCGT −28.23 TGTGTT −28.25GGGTAA −28.26 TTTTCG −28.33 GCGTTT −28.33 TCTCGG −28.34 GCGGAC −28.36CGACTT −28.38 CGACGA −28.4 GTTAGT −28.44 CCTCGC −28.53 TTGCGA −28.62GTCGCT −28.65 GTTCTG −28.7 CGCGGA −28.75 GACGTA −28.8 ATGTGT −28.81CCGCGT −28.84 TTAGGC −28.88 CTTTGG −28.94 TACCGG −29 GTAAGT −29.01ACGAGG −29.02 ACGTAG −29.02 TGGCTC −29.02 GCTTGG −29.05 ACGTTA −29.06AGGAGG −29.07 TGACGG −29.11 CCACGT −29.16 CGTATG −29.17 CGGGCA −29.21AACGGG −29.25 CTCCGG −29.27 GGGCCA −29.28 CGTACC −29.28 CCGTAC −29.41CGTACT −29.46 CTGTCT −29.48 TGCCTG −29.64 CTGTGG −29.64 TGGTTG −29.7GGTTGA −29.72 GAGGGC −29.76 TTCGGC −29.83 GGTTGC −29.89 TCTGGT −29.9CCTCGG −29.96 GTTGAC −30 TTGACG −30.03 AACGTT −30.07 CCGACC −30.12GGGTTT −30.13 GTCTAC −30.13 ACGACG −30.19 CGGGCT −30.2 GTAGAG −30.23GGAACG −30.3 GTCTTA −30.3 GCTGGC −30.31 CGTGCT −30.39 CTACGT −30.41CTTTCG −30.42 TGTCCC −30.46 CGGTAG −30.52 TCTGCG −30.54 TCGATG −30.54TCGGAC −30.58 TCCGGC −30.61 TTCGAG −30.64 CCCTCG −30.66 CCGCGA −30.67ACGTGC −30.67 GGCCTA −30.68 CCGGAC −30.7 GCGTAA −30.77 GTCCTC −30.77TTCGGA −30.82 CCCCGC −30.82 AGCGCG −30.84 CTCGCC −30.85 GGCTAG −30.87CTTACG −30.96 GATGTC −30.96 GGACGC −30.98 ACGTCT −30.99 TGTCAG −31ACGCGA −31.01 GTTCGA −31.06 TTGAGG −31.08 TCGTAC −31.09 TTAGGG −31.1TGCCGC −31.12 TAGGCC −31.12 CCCGGG −31.15 CGACCT −31.16 CGAGTC −31.3TCTGAC −31.36 GTCCCT −31.46 TCTGGG −31.48 CGCGTA −31.55 TGAGGG −31.55CGGGGA −31.59 CGACGC −31.63 TGAGGT −31.63 TTTGGC −31.64 CGTCAG −31.68GATGTG −31.69 TGGCGA −31.75 GTGAGC −31.75 GTCGTA −31.76 TCTGGC −31.78GTGTCT −31.81 GCGTCA −31.86 GCGCCT −31.88 CCTGTG −31.89 AGTCGT −31.89TCGGTA −31.95 CCGGTT −31.95 CGCGCT −31.96 CTTGGT −31.98 TTACGT −32.02GTGTAC −32.06 CGCTTG −32.07 CCGACG −32.12 CCGTGT −32.13 GTATCG −32.13TTAGTC −32.13 TCGGCC −32.14 CATGCG −32.19 GTCAGA −32.21 ACGTTG −32.23CGCATG −32.23 TCCTGT −32.37 GCGAGC −32.37 ACGTGT −32.41 ATGCGG −32.41TGGTCC −32.44 ATGGGT −32.58 CGTCTC −32.61 TGTGCC −32.64 CTTGTC −32.65GGCGGA −32.72 GTCTGA −32.74 CTGGTC −32.79 GGGGGA −32.83 AGGGTC −32.86TAGTCC −32.91 CGGGTA −32.94 GCGTTA −32.98 GACCGT −33 GATCGT −33.15ATCGGT −33.22 CACGGG −33.24 GACGTT −33.25 CACGCG −33.27 GGTAAG −33.36GTCGAT −33.37 GATGCG −33.38 GGACCG −33.38 GCCCCG −33.46 GCGGGA −33.56GGTCCC −33.6 GTATGG −33.62 CCCGTT −33.63 CGCGAT −33.69 CCGTGC −33.75GACGGT −33.89 CGACCG −33.91 CCTGTC −33.96 GTAGTG −34.01 GGGTCA −34.05TAGGGC −34.19 GTTACG −34.32 AGGTAG −34.33 GGCCGC −34.45 GCGGCC −34.5GCCTCG −34.53 CGAACG −34.71 GTCGAA −34.72 CGTCCC −34.81 CTAAGT −34.82CCGGTA −34.83 GTCTGC −34.87 TCGTGA −34.87 CGGAGT −34.91 GGGTAC −34.91GTGGAC −34.93 ACGGTC −34.94 CTCGTA −34.95 TCGAGT −35 TCTGTG −35 GGTTGT−35.09 AGGGGT −35.15 TACGTT −35.18 TCGTCA −35.34 AAGTGT −35.39 TGTAGG−35.44 GCGGTT −35.48 TACGTC −35.52 TGTTGC −35.56 TTGGTG −35.57 AGCGTG−35.58 CTGGCG −35.58 TGTACG −35.8 CGTCAT −35.87 TCCTCG −36.02 GGGCCT−36.04 CTAGTC −36.07 TGTTGG −36.07 GTGGAG −36.09 GGCCGT −36.15 GTTTGC−36.17 CCCCGT −36.18 GTGGTT −36.22 CGCCCT −36.23 TCGCCC −36.23 GATCGG−36.23 TGACCG −36.25 GGGTGA −36.29 TTCCGT −36.3 ATCGGG −36.36 TCCCGG−36.42 TGGCCA −36.53 GTGTAG −36.53 ATGCGT −36.65 GCCCGC −36.69 TGGCGC−36.74 GTGGGA −36.74 TGTTCG −36.88 TGGCCT −36.92 GGTCTA −36.94 TGCGGC−36.96 CGTGAC −37 TAACGT −37.18 TCGTTT −37.19 CGCTAG −37.2 CGGCCG −37.2CTTGCG −37.21 AGGCGG −37.21 CGTTGA −37.28 TGTTTG −37.33 GTAAGG −37.38CGGACG −37.41 CGCCGT −37.43 CGGAGG −37.44 CGTTCC −37.45 TGCGAG −37.46GTTGGT −37.56 TTTGTC −37.57 GAGGGT −37.59 TAAGTC −37.59 GGCTCG −37.63GACGCG −37.63 GGGTCT −37.66 TCGCTC −37.67 TCTCGC −37.72 TTTGTG −37.74ATCGCG −37.75 GGGGTT −37.76 GTCACG −37.82 GGTCTT −37.95 CCCGTC −37.96CTAGCG −37.99 CGCACG −38.02 TTAGGT −38.03 CGGGAG −38.06 GTTTAG −38.11GCCCGT −38.11 GTCCGA −38.11 AGTGAG −38.2 CTTGTG −38.23 TCGAGG −38.24TTGGCC −38.25 AGTCCG −38.38 CGGTTT −38.45 TGCGTT −38.48 CGTGAT −38.53GCGTTC −38.53 TTGGGG −38.54 GGTTTG −38.55 CGGTAC −38.57 TGGCCC −38.57GCTCGC −38.62 ACGCGT −38.63 TGTGAC −38.71 GACCGG −38.72 GCGCCC −38.73ACCGGG −38.81 GGTGCC −38.81 TTGTCC −38.88 TTGCCG −38.89 ACGGGT −38.92ATGTGG −39 GGTCTC −39.04 CGTAAG −39.11 TTCGTT −39.12 TACGGG −39.13GTCATG −39.17 GGACGT −39.21 CGGGAC −39.25 TGGGTT −39.32 GAGTGC −39.35CTCTCG −39.4 CGCGAC −39.42 TAGGGG −39.5 GGCACG −39.52 CCGCGC −39.55TGGACG −39.58 GGCGAC −39.6 CTGGGG −39.69 CGGGTT −39.73 GTGCCT −39.76TTGGGC −39.77 GCCGTC −39.8 GGCCAG −39.84 CCGTCC −39.9 GCGCGA −39.9CGCGGC −39.95 TCGGGA −39.98 GTCTTG −40.04 AGTGGG −40.12 CCGGGG −40.16TTTGGG −40.17 CTTCGT −40.23 CGGTCA −40.24 CACGTG −40.28 GGTGAG −40.43GTCGAC −40.48 TGCTCG −40.49 TGGGGC −40.62 GGAGGG −40.68 TGTGAG −40.75GGGGCC −40.89 GGTGGT −40.9 AGGCGT −40.91 TCCGTC −40.92 TCCGCG −40.92GTACCG −41.02 AGGTGT −41.07 GCTCGT −41.08 TTTCGT −41.14 TGCCCG −41.17CGATCG −41.22 CGTCTT −41.34 TTCCGG −41.5 GTTTTG −41.52 GCGAGT −41.58GGGGAG −41.63 CTAGGT −41.64 CCGTGG −41.64 GAGGCG −41.68 CCGCGG −41.7TTGTGC −41.74 TTTCGG −41.75 GCGTAC −41.83 GTACGC −41.88 GAGTCG −41.9TCCGTG −42.01 CGGCGA −42.01 CTCCGT −42.07 TTGCGC −42.08 GTGCCC −42.11GCGTGA −42.12 GTAGGC −42.16 CTCGTT −42.19 GTGCGA −42.23 AGTGCG −42.39CGCCCG −42.43 GGCGTA −42.43 GAGCGT −42.48 TCGTTC −42.53 AAGTCG −42.67GTCAGG −42.73 CGTTAG −42.84 TCGGTT −42.92 TCGCGA −42.92 GGGAGG −42.93GGGACG −42.94 GTCAGT −43.2 TGCCGT −43.2 GGGGTA −43.2 GCGTCT −43.23GCCGCG −43.26 AGTCGG −43.28 TCCGTT −43.36 CTCGGG −43.37 GGGCCC −43.5TAGGCG −43.53 GGTCAG −43.58 GGGTAG −43.61 TACGTG −43.67 GTCCTG −43.69CGCCGC −43.8 CTGGCC −43.85 TAGGGT −43.88 GCCGGG −43.92 GGTTAG −43.96CCGGGT −44.07 CTCGTC −44.16 GTCTAG −44.19 GGTGTT −44.21 CCGGGC −44.24AGTGTG −44.25 CGAGGG −44.3 GTTGGC −44.3 CGGCGC −44.3 AGTGTC −44.31CGTTGT −44.33 GTTCGC −44.35 GTTGCC −44.36 GGCGGT −44.48 TTCGCG −44.51TTGCGG −44.57 GTGTTC −44.64 ATGTCG −44.73 GGCGGC −44.81 TCGGAG −44.82GACGGG −44.82 CGTCCT −44.86 TCGACG −44.94 GGACGG −44.99 TGGTGG −44.99TCCCGT −45.06 TGTCGA −45.08 GCTCGG −45.1 GGGCCG −45.15 GTTTGT −45.27GAGGGG −45.32 TTCGTG −45.45 GCGAGG −45.47 CCTCGT −45.53 GCCCGG −45.6GTCTGT −45.62 TTGTCT −45.66 CGGTGT −45.71 CGTTTG −45.74 GGTAGG −45.84GTCCGC −45.88 GCCGGC −45.88 CGCGTT −45.93 ACGGGG −45.94 CCGTTG −45.97TCTGTC −46.05 GGGCGA −46.06 GACGTG −46.08 TTGGTC −46.16 GCCGGT −46.32TTGCGT −46.32 GGGTTG −46.34 GGCGAG −46.43 CGTGTT −46.44 GGGTCC −46.51TGGGCC −46.53 GCGTTG −46.58 CGACGG −46.68 AGGGGG −46.69 GTGTCA −46.75GCGTAG −46.76 TAGGTC −46.77 CGCGAG −46.79 TGAGTG −47.04 GACGTC −47.04GTCGGA −47.14 GGTTGG −47.18 TCGCGC −47.26 GCGCCG −47.28 TGTCTG −47.32GCCGTG −47.35 CGTTGC −47.38 TCGTCT −47.39 GGCGCC −47.47 GGGGGC −47.53TTGTGG −47.6 CGGTCC −47.61 CGCGCC −47.71 TTCGGT −47.79 TGACGT −47.8TGTCCG −47.88 TGTTGT −47.91 CCCGGT −48.15 GCGTCC −48.17 TCCGGG −48.25CCCGCG −48.5 TCGTCC −48.56 GTTCGT −48.56 GTTCCG −48.59 TTGGCG −48.69TGGCCG −48.69 GCGACG −48.74 GGAGTG −48.78 GTTAGG −49.18 GGCCGG −49.22CGGTTG −49.22 TCTCGT −49.23 CGAGCG −49.24 CGAGGT −49.43 CGTCTG −49.43CTCGGT −49.55 TTCGTC −49.6 GGCGTT −49.72 TCGGCG −49.79 CGTCGA −49.86GTGACG −49.87 CGACGT −49.9 GGTACG −49.96 CGGTGC −50.01 GTACGG −50.02CGTGAG −50.26 CGGCGG −50.36 TTGTGT −50.37 GAGTGG −50.58 TTCGGG −50.66TGTGTC −50.68 TGGGTC −50.7 GGTCGA −50.8 GTTTGG −50.88 CCCGTG −51.09GTTTCG −51.17 CGAGTG −51.21 GAGTGT −51.21 TGGTGT −51.29 TCGGGC −51.42TGCGCG −51.46 TCGCCG −51.51 CCGGTC −51.68 CGCGTC −51.71 GTCGTT −51.72TGGTCT −51.83 CGCGGT −51.85 GGTCTG −51.86 CTCGCG −51.88 CTCGTG −51.94CGGGCC −52.44 GTACGT −52.73 AGGGCG −52.77 GTGCCG −52.81 GTGAGT −52.89TGTGGT −52.99 CGGTCT −53.11 TCGTGG −53.14 CGGGGC −53.26 TCGTTG −53.27ACGTGG −53.35 GGTTCG −53.38 ACGTCG −53.48 GGCCCG −53.53 CGTGCC −53.55TGGGGG −53.57 CGGCGT −53.63 CGTAGG −53.63 GTCTGG −53.69 GTGAGG −53.7CGTACG −53.71 GTTGTC −53.73 TTGGGT −53.74 CGTCCG −53.8 TGCCGG −53.82TCGTGC −53.92 CGGGTC −54 GTCGAG −54.01 CGTTGG −54.19 CCGGCG −54.27TCCGGT −54.32 GCGGGT −54.37 TCGTGT −54.38 CGCCGG −54.53 CGCTCG −54.55GTCCGT −54.62 GGTGGC −54.7 TGCGTC −54.83 GGGTGC −54.96 GTCGCC −55.39TGTGCG −55.49 CGTGTC −55.5 GGCGTC −55.61 GCGCGC −55.66 CTGTCG −55.74GTCCCG −56.36 GCGGGC −56.49 GTAGGG −56.76 TGTCGC −56.8 TCGCGG −56.94TGGCGT −57.03 GTGCGC −57.04 TTGTCG −57.09 GTGTTG −57.15 TGGGGT −57.19GGTCGC −57.25 CGTGGC −57.9 GGGCGC −58.19 TGCGGT −58.27 TGGCGG −58.3GGGGGT −58.38 TCGGGT −58.51 CCGGTG −58.6 CGTTCG −58.67 TCGGTC −58.82GTCGGC −58.88 GTGGTC −58.88 GTGTGA −59.14 CGTGGT −59.24 GTGGCC −59.29GCGGTC −59.3 GCGCGT −59.36 AGGTCG −59.5 GTCTCG −59.51 GGTGTC −59.7TGGTCG −59.72 GCGGTG −60.02 TGCGTG −60.04 GTGTGT −60.05 GGGGTC −60.15CGCGCG −60.19 TGGGCG −60.25 GCGTGT −60.27 GTTGGG −60.36 TGCGGG −60.39TGTGGC −60.71 GCGCGG −60.73 CGTCGC −60.8 CCGTCG −60.85 GTGGTG −60.86GTTCGG −61.52 GGGCGG −61.53 TCGCGT −61.64 GTGTCC −61.73 GGGTGT −61.79GGGGGG −62.06 TGTGTG −62.08 GCGTGC −62.32 CGGGGG −62.44 CGGGCG −62.52GGCGTG −62.89 TCGGTG −63.03 GGCGGG −63.07 GTTGTG −63.22 GGTCGT −63.3TCGGGG −63.6 GTTGCG −64.3 GGGCGT −64.62 TCGTCG −64.83 GGTCCG −64.88GCGGCG −64.99 GTGCGG −65.11 GGTGCG −65.21 GCGTGG −65.85 GGGGCG −66.57CGCGTG −66.73 GTGTGC −66.98 GTCCGG −67.1 GTGCGT −67.14 TGTCGT −67.26TGTGGG −67.31 CGGTCG −67.35 CGGGGT −67.36 CGCGGG −67.6 TGTCGG −67.61CGTCGG −68.18 GGCGCG −68.24 GGGGTG −68.68 CGTCGT −68.69 GTCGGT −68.84TGGGTG −69.08 GTCGTC −69.14 GCGTCG −69.26 CGGGTG −69.69 GGGTGG −69.98GTGGGC −70.27 CGTGTG −71.38 CGGTGG −71.52 CGTGCG −71.83 GCGGGG −72.46GTGGGT −73.21 GTCGCG −73.55 GTCGTG −73.94 GTGGCG −73.94 GTGGGG −74.96GGTGGG −75.37 CGTGGG −75.74 GGGTCG −76.6 GTCGGG −80.38 GGTCGG −81.93GGTGTG −82.57 GTGTCG −84.85 GTGTGG −90.52

As one of skill in the art will appreciate, the rank ordering of the SHMmotifs described above provides for a method whereby synthetic geneconstructs can be created that are more susceptible to SHM relative to astarting sequence by the replacement of any specific SHM motif with onethat has a greater probability of SHM mediated mutagenesis. Converselysynthetic gene constructs can be created that are more resistant to SHMrelative to a starting sequence by the replacement of any specific SHMmotif with one that has a lower probability of SHM mediated mutagenesis.

In certain embodiments, polynucleotide motifs having rank-orderedz-scores in the top 5% of all equivalent length polynucleotide motifscan be considered SHM “hot spots,” and can be inserted into a gene tomake a polynucleotide sequence more SHM susceptible. In certain otherembodiments, polynucleotide motifs having rank-ordered z-scores in thetop 10% of all equivalent length polynucleotide motifs can be consideredSHM “hot spots,” and can be inserted into a gene to make apolynucleotide sequence more SHM susceptible. In still otherembodiments, polynucleotide motifs having rank-ordered z-scores in thetop 15% of all equivalent length polynucleotide motifs can be consideredSHM “hot spots,” and can be inserted into a gene to make apolynucleotide sequence more SHM susceptible. In yet other embodiments,polynucleotide motifs having rank-ordered z-scores in the top 20% of allequivalent length polynucleotide motifs can be considered SHM “hotspots,” and can be inserted into a gene to make a polynucleotidesequence more SHM susceptible. In yet still other embodiments,polynucleotide motifs having rank-ordered z-scores in the top 25% of allequivalent length polynucleotide motifs can be considered SHM “hotspots,” and can be inserted into a gene to make a polynucleotidesequence more SHM susceptible.

Likewise, polynucleotide motifs having rank-ordered z-scores in thebottom 5% of all equivalent length polynucleotide motifs can beconsidered SHM “cold spots,” and can be inserted into a gene to make apolynucleotide sequence more SHM resistant. In other embodiments,polynucleotide motifs having rank-ordered z-scores in the bottom 10% ofall equivalent length polynucleotide motifs can be considered SHM “coldspots,” and can be inserted into a gene to make a polynucleotidesequence more SHM resistant. In still other embodiments, polynucleotidemotifs having rank-ordered z-scores in the bottom 15% of all equivalentlength polynucleotide motifs can be considered SHM “cold spots,” and canbe inserted into a gene to make a polynucleotide sequence more SHMresistant. In yet other embodiments, polynucleotide motifs havingrank-ordered z-scores in the bottom 20% of all equivalent lengthpolynucleotide motifs can be considered SHM “cold spots,” and can beinserted into a gene to make a polynucleotide sequence more SHMresistant. In yet still other embodiments, polynucleotide motifs havingrank-ordered z-scores in the bottom 25% of all equivalent lengthpolynucleotide motifs can be considered SHM “cold spots,” and can beinserted into a gene to make a polynucleotide sequence more SHMresistant.

The position or reading frame of a hot spot or cold spot is also animportant factor governing whether SHM mediated mutagenesis that canresult in a mutation that is silent with regards to the resulting aminoacid sequence, or causes conservative, semi-conservative or nonconservative changes at the amino acid level. As discussed below, thesedesign parameters can be manipulated to further enhance the relativesusceptibility or resistance of a nucleotide sequence to SHM.

Thus both the degree of SHM recruitment and the reading frame of themotif are considered in the design of SHM susceptiable and SHM resistantpolynucleotide sequences.

An optimized polynucleotide sequence has been made “susceptible for SHM”or “hot” if the polynucleotide sequence, or a portion thereof, has beenaltered, or designed, to increase the frequency and/or location of hotspots within the open reading frame and/or has been altered, ordesigned, to decrease the frequency and/or location of cold spots withinthe open reading frame of the polynucleotide sequence compared to thewild type polynucleotide sequence.

Conversely, an optimized polynucleotide sequence has been made“resistant to SHM” or “cold” if the polynucleotide sequence; or aportion thereof, has been altered to decrease the frequency and/orlocation of hot spots within the open reading frame of thepolynucleotide sequence, and/or has been altered, or designed, toincrease the frequency and/or location of cold spots within the openreading frame of the polynucleotide sequence compared to the wild typepolynucleotide sequence.

Provided herein is a strategy to design nucleotide templates to eithermaximize or minimize the tendency of a polynucleotide to undergo SHM,while at the same time maximizing protein expression, RNA stability, andthe presence of conveniently located restriction enzyme sites.

Also provided herein are synthetic versions of a polynucleotide that arealtered to either enhance, or decrease the impact of SHM on the rate ofmutagenesis of that polynucleotide compared to its wild type'ssusceptibility to undergo SHM (i.e., SHM susceptible or SHM resistent).

Also provided herein are synthetic versions of a polynucleotide in whichspecific regions of a polynucleotide have been optimized to be eitherSHM resistant or SHM susceptible. In one embodiment, functional portionand/or regions of a polynucleotide can be hot (e.g., ligand binding,enzymatic activity, etc.) while other regions (e.g., those needed forstructural folding, conformation, etc.) of a polynucleotide can be madecold.

The SHM susceptible sequences facilitate the rapid evolution andselection of improved mutant versions of proteins and the systemcombines the power of rational design with accelerated randommutagenesis and directed evolution.

Also included in the invention are SHM resistant polynucleotidesequences that allow for conserved regions to be resistant toSHM-mediated mutagenesis, while simultaneously targeting desiredsequences for increased susceptibility to SHM-mediated mutagenesis. Thusit is possible to optimize particular functional portions and/or regionsof a polynucleotide that appear to be directly involved in a functionalattribute of a protein encoded by the polynucleotide.

In one non-limiting example, nucleotides to be optimized can encodeamino acids that can lie within, or within about 5 Å of a specificfunctional or structural attribute of interest. Specific examples offunctional portions and/or regions include, but are not limited to,amino acids within CDRs of antibodies, binding pockets of receptors,catalytic clefts of enzymes, protein-protein interaction domains, ofco-factors, allosteric binding sites, etc.

Polynucleotides for which these methods are applicable include anypolynucleotide sequence that can be transcribed and a functional assaydevised for screening. Preferred polynucleotide sequences include thoseencoding proteins, polypeptides and peptides such as, for example,specific binding members, antibodies or fragment thereof, an antibodyheavy chain or portion thereof, an antibody light chain or portionthereof, an intrabodies, selectable marker genes, enzymes, receptors,peptide growth factors and hormones, co-factors, and toxins.

Other non-limiting examples of molecules for use herein includepolynucleotides that have enzymatic or binding activity without the needfor translation into a protein or peptide sequence, such polynucleotidesincluding for example, enzymatic nucleic acids, antisense nucleic acids,triplex forming oligonucleotides, 2,5-A chimeras, RsiNA, dsRNA,allozymes, abd aptamers.

Biologically active molecules of the invention also include moleculescapable of modulating the pharmacokinetics and/or pharmacodynamics ofother biologically active molecules, for example, lipids and polymerssuch as polyamines, polyamides, polyethylene glycol and otherpolyethers. For example, polypeptides are those such as, for example,VEGF, VEGF receptor, Diptheria toxin subunit A, B. pertussis toxin, CCchemokines (e.g., CCL1-CCL28), CXC chemokines (e.g., CXCL1-CXCL16), Cchemokines (e.g., XCL1 and XCL2) and CX3C chemokines (e.g., CX3CL1),IFN-gamma, IFN-alpha, IFN-beta, TNF-alpha, TNF-beta, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, TGF-beta, TGF-alpha,GM-CSF, G-CSF, M-CSF, TPO, EPO, human growth factor, fibroblast growthfactor, nuclear co-factors, Jak and Stat family members, G-proteinsignaling molecules such as chemokine receptors, JNK, Fos-Jun, NF-κB,I-κB, CD40, CD4, CD8, B7, CD28 and CTLA-4.

IV. Strategies for Designing Polynucleotide Sequences that are SHMResistant or SHM Susceptible

The design and use of SHM optimized sequences is described in priorityU.S. application No. 60/902,414.

One strategy for altering the ability of a polynucleotide to undergo SHMis through altering the codon usage to modulate SHM hot spot and/or coldspot density, this approach enables hot spot density to be increased ordecreased without impact on the primary amino acid sequence of theprotein of interest.

In addition to optimizing hot spot and/or cold spot density, it is alsodesirable to consider the following characteristics such that theoptimized polynucleotides are efficiently translated, and stable in ahost system. As discussed below, these design parameters can beconveniently optimized using an iterative computer algorithm.

The density of CpG dinucleotides motifs: Excessive CG motifs can resultin gene methylation leading to gene silencing, and can be normalized tothe density found in highly transcribed gene in the host system inquestion (see for example, Kameda et al., Biochem. Biophys. Res. Commun.(2006) 349(4): 1269-1277).

The ability of single stranded sequences to form stem-loop structures:the formation of stem-loop structures can result inefficienttranscription and or translation, particularly when located near the 5′region of the coding frame (see, e.g., Zuker M., Mfold web server fornucleic acid folding and hybridization prediction. Nucl. Acid Res.(2003); 31(13): 3406-3415). Stem loop structure formation can beminimized by avoiding repetitive or palindromic stretches of greaterthan 6 nucleotides, for example, near the 5′ end. Alternatively, longerstems are acceptable if the loop contains greater than about 25nucleotides (nt).

Codon Usage: Appropriate codon usage, i.e., the use of codons thatencode for more common and frequently used tRNAs, rather than very raretRNAs, is important to enable efficient translation in the expressionsystem being used (see generally Nakamura et al., Nuc. Acid. Res. (2000)28 (1): 292, “Codon usage tabulated from international DNA sequencedatabases: status for the year 2000;” which includes codon frequencytables of each of the complete protein sequences in the GenBank DNAsequence database as of 2000). Generally codon usage is more importantnear the 5′ end of the gene where transcription of the polynucleotidebegins and rare codons should be avoided in this region where everpossible. Preferred is the elimination of about 80% or more of thecodons that are used less than 10% of the time within the coding frameof the expressed genes in the organism of interest.

GC content: Generally this should be matched, to the GC content ofhighly expressed genes in the host organism, for example in mammaliansystems GC content should be less than about 60%.

Restriction sites: Restriction sites should be placed judiciously wheredesired. Similarly, important restriction sites (i.e. those that areintended to be used to clone the entire gene, or other genes) within apolynucleotide should be removed where not desired by altering wobblepositions.

Stretches of the same nucleotide: Minimize or eliminate stretches of thesame nucleotide to less six (6) contiguous nucleotides.

In addition, expression can be further optimized by including a Kozakconsensus sequence [i.e., (a/g)cc(a/g)ccATGg] at the start codon. Kozakconsensus sequences useful for this purpose are known in the art (Mantyhet al. PNAS 92: 2662-2666 (1995); Mantyh et al. Prot. Exp. & Purif.6,124 (1995)).

Non-preferred codon usage: Avoid or minimize the usage of certain codons(“non preferred SHM codons”) that can be mutated in one step to create astop codon. “Non preferred codons” include, UGG (Trp), UGC (Cys), UCA(Ser), UCG (Ser), CAA, (Q) GAA (Glu) and CAG (Gln).

Beyond sequence specific constraints within the coding sequence of thepolynucleotide of interest, additional design criteria for engineering apolynucleotide sequence with altered susceptibility to SHM includes thefollowing factors:

The choice of promoter; a strong promoter will generally induce a higherrate of transcription resulting a higher overall rate of mutagenesiscompared to a weaker promoter. Further, an inducible promoter, such asthe tet-promoter enables expression, and hence SHM, to be induciblycontrolled, to switch on, or off, transcription and mutagenesis of thepolynucleotide of interest. Gossen and Bujard, Tight control of geneexpression in mammalian cells by tetracycline-responsive promoters. ProcNatl Acad Sci USA. 1992 Jun. 15; 89(12):5547-51; Gossen et al.,Transcriptional activation by tetracyclines in mammalian cells. Science.1995 Jun. 23; 268(5218):1766-9.

The location of the coding sequence relative to the transcriptionalstart point; generally for high level mutagenesis, the polynucleotide ofinterest should be located between about 50 nucleotides, and 2 kb of thetranscriptional start site.

One convenient approach to optimizing a polynucleotide sequence to SHM,involves analyzing the corresponding amino acid sequence of interest viaa computer algorithm that compares and scores (according to theparameters above) possible alternative polynucleotides sequences thatcan be used, via alternative codon usage to encode for the amino acidsequence of interest. By iteratively replacing codons, or groups ofcodons (tiles, or SHM motifs) with progressively preferred sequences itis possible to computationally evolve a polynucleotide sequence withdesired properties. Specifically, for example, a sequence that is SHMsusceptiable, or that is resistant to SHM, and yet also exhibitsreasonable translational efficiency, stability, minimizes restrictionsites and avoids rare codons in the particular organism of interest.

Using this approach, a library of files can be generated that is basedon the starting amino acid or polynucleotide sequence. In one nonlimiting example of the analysis and optimization strategy, the librarycan be created based on the analysis of groups of 9 nucleotides,corresponding to 3 codons (a “tile”). Each tile can be scored for theattributes described above, to create an initial library data set oftiles, containing hundreds of thousands of 9-mer permutations, and theirrespective scores.

A representative sample of a section of the library file is shown inTable 4 which shows the potential diversity in nucleotide sequencesarising from alternative codon usage for just the three amino acids,Serine (S), Arginine (R) and Leucine (L). A person of skill in the artreadily appreciates that a complete set of files can be readilyassembled for all possible amino acid combinations using known codonusage patterns.

TABLE 4 Representative polynucleotide diversity encoding a three aminoacid sequence (Ser Arg Leu) 3-mer Potential SEQ AA nucleotides ID NOHotspots Coldspots CpG MaxNt Log(πp(AA)) SRL AGTCGACTT 68 0 2 1 1 −5 SRLAGTCGACTG 69 0 2 1 1 −3 SRL AGTCGATTA 70 0 1 1 2 −5 SRL AGTCGACTA 71 0 21 1 −5 SRL AGTCGACTC 72 0 3 1 1 −4 SRL AGTCGATTG 73 0 1 1 2 −5 SRLAGTAGGCTG 74 2 0 0 2 −4 SRL AGTAGGCTG 75 2 0 0 2 −2 SRL AGTAGGTTA 76 2 00 2 −4 SRL AGTAGGCTA 77 2 0 0 2 −4 SRL AGTAGGCTC 78 2 1 0 2 −3 SRLAGTAGGTTG 79 2 0 0 2 −4 SRL AGTCGTCTT 80 0 2 1 1 −5 SRL AGTCGTCTG 81 0 21 1 −3 SRL AGTCGTTTA 82 0 1 1 3 −5 SRL AGTCGTCTA 83 0 2 1 1 −5 SRLAGTCGTCTC 84 0 3 1 1 −4 SRL AGTCGTTTG 85 0 1 1 3 −5 SRL AGTAGACTT 86 1 10 1 −4 SRL AGTAGACTG 87 1 1 0 1 −2 SRL AGTAGATTA 88 1 0 0 2 −4 SRLAGTAGACTA 89 1 1 0 1 −4 SRL AGTAGACTC 90 1 2 0 1 −3 SRL AGTAGATTG 91 1 00 2 −4 SRL AGTCGGCTT 92 1 1 1 2 −4 SRL AGTCGGCTG 93 1 1 1 2 −2 SRLAGTCGGTTA 94 1 1 1 2 −4 SRL AGTGGGCTA 95 1 1 1 2 −4 SRL AGTCGGCTC 96 1 21 2 −3 SRL AGTCGGTTG 97 1 1 1 2 −4 SRL AGTCGCCTT 98 0 2 1 2 −4 SRLAGTCGCCTG 99 0 2 1 2 −2 SRL AGTCGCTTA 100 0 1 1 2 −4 SRL AGTCGCCTA 101 02 1 2 −4 SRL AGTCGCCTC 102 0 3 1 2 −3 SRL AGTCGCTTG 103 0 1 1 2 −4 SRLTCACGACTT 104 0 1 1 1 −5 SRL TCACGACTG 105 0 1 1 1 −3 SRL TCACGATTA 1060 0 1 2 −5 SRL TCACGACTA 107 0 1 1 1 −5 SRL TCACGACTC 108 0 2 1 1 −4 SRLTCACGATTG 109 0 0 1 2 −5 SRL TCAAGGCTT 110 1 0 0 2 −4 SRL TGAAGGCTG 1111 0 0 2 −2 SRL TCAAGGTTA 112 1 0 0 2 −4 SRL TCAAGGCTA 113 1 0 0 2 −4 SRLTCAAGGCTC 114 1 1 0 2 −3 SRL TCAAGGTTG 115 1 0 0 2 −4 SRL TCACGTCTT 1160 1 1 1 −5 SRL TCACGTCTG 117 0 1 1 1 −3 SRL TCACGTTTA 118 0 0 1 3 −5 SRLTCACGTCTA 119 0 1 1 1 −5 SRL TCACGTCTC 120 0 2 1 1 −4 SRL TCACGTTTG 1210 0 1 3 −5 SRL TCAAGACTT 122 0 1 0 2 −4 SRL TCAAGACTG 123 0 1 0 2 −2 SRLTCAAGATTA 124 0 0 0 2 −4 SRL TCAAGACTA 125 0 1 0 2 −4 SRL TCAAGACTC 1260 2 0 2 −3 SRL TCAAGATTG 127 0 0 0 2 −4 SRL TCACGGCTT 128 1 0 1 2 −4 SRLTCACGGCTG 129 1 0 1 2 −2 SRL TCACGGTTA 130 1 0 1 2 −4 SRL TCACGGCTA 1311 0 1 2 −4 SRL TCACGGCTC 132 1 1 1 2 −3 SRL TCACGGTTG 133 1 0 1 2 −4 SRLTCACGCCTT 134 0 1 1 2 −4 SRL TCACGCCTG 135 0 1 1 2 −2 SRL TCACGCTTA 1360 0 1 2 −4 SRL TCACGCCTA 137 0 1 1 2 −4 SRL TCACGCCTC 138 0 2 1 2 −3 SRLTCACGCTTG 139 0 0 1 2 −4 SRL AGCCGACTT 140 1 2 1 2 −5 SRL AGCCGACTG 1411 2 1 2 −3 SRL AGCCGATTA 142 1 1 1 2 −5 SRL AGCCGACTA 143 1 2 1 2 −5 SRLAGCCGACTC 144 1 3 1 2 −4 SRL AGCCGATTG 145 1 1 1 2 −5 SRL AGCAGGCTT 1462 0 0 2 −4 SRL AGCAGGCTG 147 2 0 0 2 −2 SRL AGCAGGTTA 148 2 0 0 2 −4 SRLAGCAGGCTA 149 2 0 0 2 −4 SRL AGCAGGCTC 150 2 1 0 2 −3 SRL AGCAGGTTG 1512 0 0 2 −4 SRL AGCCGTCTT 152 1 2 1 2 −5 SRL AGCCGTCTG 153 1 2 1 2 −3 SRLAGCCGTTTA 154 1 1 1 3 −5 SRL AGCCGTCTA 155 1 2 1 2 −5 SRL AGCCGTCTC 1561 3 1 2 −4 SRL AGCCGTTTG 157 1 1 1 3 −5 SRL AGCAGACTT 158 1 1 0 1 −4 SRLAGCAGACTG 159 1 1 0 1 −2 SRL AGCAGATTA 160 1 0 0 2 −4 SRL AGCAGACTA 1611 1 0 1 −4 SRL AGCAGACTC 162 1 2 0 1 −3 SRL AGCAGATTG 163 1 0 0 2 −4 SRLAGCCGGCTT 164 2 1 1 2 −4 SRL AGCCGGCTG 165 2 1 1 2 −2 SRL AGCCGGTTA 1662 1 1 2 −4 SRL AGCCGGCTA 167 2 1 1 2 −4 SRL AGCCGGCTC 168 2 2 1 2 −3 SRLAGCCGGTTG 169 2 1 1 2 −4 SRL AGCCGCCTT 170 1 2 1 2 −4 SRL AGCCGCCTG 1711 2 1 2 −2 SRL AGCCGCTTA 172 1 1 1 2 −4 SRL AGCCGCCTA 173 1 2 1 2 −4 SRLAGCCGCCTC 174 1 3 1 2 −3 SRL AGCCGCTTG 175 1 1 1 2 −4 SRL TCGCGACTT 1760 1 2 1 −6 SRL TCGCGACTG 177 0 1 2 1 −4 SRL TCGCGATTA 178 0 0 2 2 −6 SRLTCGCGACTA 179 0 1 2 1 −6 SRL TCGCGACTC 180 0 2 2 1 −5 SRL TCGCGATTG 1810 0 2 2 −6 SRL TCGAGGCTT 182 1 1 1 2 −5 SRL TCGAGGCTG 183 1 1 1 2 −3 SRLTCGAGGTTA 184 1 1 1 2 −5 SRL TCGAGGCTA 185 1 1 1 2 −5 SRL TCGAGGCTC 1861 2 1 2 −4 SRL TCGAGGTTG 187 1 1 1 2 −5 SRL TCGCGTCTT 188 0 1 2 1 −6 SRLTCGCGTCTG 189 0 1 2 1 −4 SRL TCGCGTTTA 190 0 0 2 3 −6 SRL TCGCGTCTA 1910 1 2 1 −6 SRL TCGCGTCTC 192 0 2 2 1 −5 SRL TCGCGTTTG 193 0 0 2 3 −6 SRLTCGAGACTT 194 0 2 1 1 −5 SRL TCGAGACTG 195 0 2 1 1 −3 SRL TCGAGATTA 1960 1 1 2 −5 SRL TCGAGACTA 197 0 2 1 1 −5 SRL TCGAGACTC 198 0 3 1 1 −4 SRLTCGAGATTG 199 0 1 1 2 −5 SRL TCGCGGCTT 200 1 0 2 2 −5 SRL TCGCGGCTG 2011 0 2 2 −3 SRL TCGCGGTTA 202 1 0 2 2 −5 SRL TCGCGGCTA 203 1 0 2 2 −5 SRLTCGCGGCTC 204 1 1 2 2 −4 SRL TCGCGGTTG 205 1 0 2 2 −5 SRL TCGCGCCTT 2060 1 2 2 −5 SRL TCGCGCCTG 207 0 1 2 2 −3 SRL TCGCGCTTA 208 0 0 2 2 −5 SRLTCGCGCCTA 209 0 1 2 2 −5 SRL TCGCGCCTC 210 0 2 2 2 −4 SRL TCGCGCTTG 2110 0 2 2 −5 SRL TCCCGACTT 212 0 2 1 3 −5 SRL TCCCGACTG 213 0 2 1 3 −3 SRLTCCCGATTA 214 0 1 1 3 −5 SRL TCCCGACTA 215 0 2 1 3 −5 SRL TCCCGACTC 2160 3 1 3 −4 SRL TCCCGATTG 217 0 1 1 3 −5 SRL TCCAGGCTT 218 1 0 0 2 −4 SRLTCCAGGCTG 219 1 0 0 2 −2 SRL TCCAGGTTA 220 1 0 0 2 −4 SRL TCCAGGCTA 2211 0 0 2 −4 SRL TCCAGGCTC 222 1 1 0 2 −3 SRL TCCAGGTTG 223 1 0 0 2 −4 SRLTCCCGTCTT 224 0 2 1 3 −5 SRL TCCCGTCTG 225 0 2 1 3 −3 SRL TCCCGTTTA 2260 1 1 3 −5 SRL TCCCGTCTA 227 0 2 1 3 −5 SRL TCCCGTCTC 228 0 3 1 3 −4 SRLTCCCGTTTG 229 0 1 1 3 −5 SRL TCCAGACTT 230 0 1 0 2 −4 SRL TCCAGACTG 2310 1 0 2 −2 SRL TCCAGATTA 232 0 0 0 2 −4 SRL TCCAGACTA 233 0 1 0 2 −4 SRLTCCAGACTC 234 0 2 0 2 −3 SRL TCCAGATTG 235 0 0 0 2 −4 SRL TCCCGGCTT 2361 1 1 3 −4 SRL TCCCGGCTG 237 1 1 1 3 −2 SRL TCCCGGTTA 238 1 1 1 3 −4 SRLTCCCGGCTA 239 1 1 1 3 −4 SRL TCCCGGCTC 240 1 2 1 3 −3 SRL TCCCGGTTG 2411 1 1 3 −4 SRL TCCCGCCTT 242 0 2 1 3 −4 SRL TCCCGCCTG 243 0 2 1 3 −2 SRLTCCCGCTTA 244 0 1 1 3 −4 SRL TCCCGCCTA 245 0 2 1 3 −4 SRL TCCCGCCTC 2460 3 1 3 −3 SRL TCCCGCTTG 247 0 1 1 3 −4 SRL TCTCGACTT 248 0 2 1 1 −5 SRLTCTCGACTG 249 0 2 1 1 −3 SRL TCTCGATTA 250 0 1 1 2 −5 SRL TCTCGACTA 2510 2 1 1 −5 SRL TCTCGACTC 252 0 3 1 1 −4 SRL TCTCGATTG 253 0 1 1 2 −5 SRLTCTAGGCTT 254 1 0 0 2 −4 SRL TCTAGGCTG 255 1 0 0 2 −2 SRL TCTAGGTTA 2561 0 0 2 −4 SRL TCTAGGCTA 257 1 0 0 2 −4 SRL TCTAGGCTC 258 1 1 0 2 −3 SRLTCTAGGTTG 259 1 0 0 2 −4 SRL TCTCGTCTT 260 0 2 1 1 −5 SRL TCTCGTCTG 2610 2 1 1 −3 SRL TCTCGTTTA 262 0 1 1 3 −5 SRL TCTCGTCTA 263 0 2 1 1 −5 SRLTCTCGTCTC 264 0 3 1 1 −4 SRL TCTCGTTTG 265 0 1 1 3 −5 SRL TCTAGACTT 2660 1 0 1 −4 SRL TCTAGACTG 267 0 1 0 1 −2 SRL TCTAGATTA 268 0 0 0 2 −4 SRLTCTAGACTA 269 0 1 0 1 −4 SRL TCTAGACTC 270 0 2 0 1 −3 SRL TCTAGATTG 2710 0 0 2 −4 SRL TCTCGGCTT 272 1 1 1 2 −4 SRL TCTCGGCTG 273 1 1 1 2 −2 SRLTCTCGGTTA 274 1 1 1 2 −4 SRL TCTCGGCTA 275 1 1 1 2 −4 SRL TCTCGGCTC 2761 2 1 2 −3 SRL TCTCGGTTG 277 1 1 1 2 −4 SRL TCTCGCCTT 278 0 2 1 2 −4 SRLTCTCGCCTG 279 0 2 1 2 −2 SRL TCTCGCTTA 280 0 1 1 2 −4 SRL TCTCGCCTA 2810 2 1 2 −4 SRL TCTCGCCTC 282 0 3 1 2 −3 SRL TCTCGCTTG 283 0 1 1 2 −4

Each polynucleotide sequence is ranked based on the followingattributes; number of SHM hot and cold motifs, number of CpG motifs,MaxNt (maximum number of nucleotides in a single stretch) and codonusage frequency of the host cell to be used. The term “Log(πp(AA)”contained in the final column of Table 4 was calculated as the log ofthe product of the individual probabilities of observing each of theamino acids in the trimer, given by the formula:

Log(πp(AA)=ln(p(codon_(i−1)|amino acid_(i−1))*p(codon_(i)|aminoacid_(i))*p(codon_(i+i)|amino acid_(i+1)).

Individual probabilities for each amino acid were based on publishedcodon usage patterns in the organism of interest, in this case, formammalian cells. (See generally Nakamura et al., Nucleic Acid Res.(2000) 28 (1): 292 Codon usage tabulated from international DNA sequencedatabases: status for the year 2000).

As can be readily seen from the Table above, codon usage diversity aloneenables polynucleotide sequences to be created that vary widely in theirsusceptibility to somatic hypermutation, as measured by the number ofhot or cold spots present within the sequence.

This analysis readily identifies potential combinations of codons thatare optimized for SHM and minimize CpGs and use optimal codons forefficient translation. For example, the sequences listed below representtop ranking hot sequences because they comprise the maximum number ofhot spots and no cold spots.

TABLE 5 Top Hot Spot Sequences SEQ 3-mer Potential ID Hot Cold Max LogAA nucleotides NO Spots Spots CpG Nt (np(AA)) SRL AGTAGGCTT 284. 2 0 0 2−4 SRL AGTAGGCTG 285. 2 0 0 2 −2 SRL AGTAGGTTA 286. 2 0 0 2 −4 SRLAGTAGGCTA 287. 2 0 0 2 −4 SRL AGCAGGCTT 288. 2 0 0 2 −4 SRL AGCAGGCTG289. 2 0 0 2 −2 SRL AGCAGGTTA 290. 2 0 0 2 −4 SRL AGCAGGCTA 291. 2 0 0 2−4 SRL AGCAGGTTG 292. 2 0 0 2 −4 SRL AGTAGGTTG 293. 2 0 0 2 −4

Of these, the sequences AGTAGGCTG (SEQ ID NO: 285) and AGCAGGCTG (SEQ IDNO: 289) are preferred because they encompass codons with a higherfrequency of use in mammalian cells.

Having defined and scored all possible 9-mer nucleotide tiles, it ispossible to scan through a starting amino acid or nucleotide template,identifying positions in the gene/protein that can be improved bysubstitution from the tile library. This process can be convenientlycompleted using a computer algorithm, such as the per1 programSHMredesign.p1; the code of which is shown below:

#! /usr/bin/perl ############ # #  by PM Bowers #  Apr 15th 2006#  Anaptys Biosciences Inc. # #  A program to redesign protein andnucleic acid sequences to be hot or cold to SHM # ############################################################################################################## # #  Read in the genetic code, amino acids, andmammalian codon usage frequencies # #  $cod_aa{ } -> mapping of codon toamino acid #  $cod_anti{ } -> mapping of codon to its opposite strandsequence #  $codnum{ } -> frequency per 1000 of observing the codon inmammals #  $tot_cod{ } -> frequency per 1000 of observing that codon inmammals, given the identity of the amino acid #  $aa_cod{ }{ } -> mapsan amino acid to its codons with the frequency found in mammalian genes################################################################################################# open(GENE, .”/geneticcode.txt”); while (<GENE>) {    if(/{circumflex over ( )}(\S+)\s+(\S+)\s+(\S+)\s+(\S+)\t(\d+)\t(\d+)/) {$one=$1; $four=$4; $five=$5; $six=$6; $thr=$3;      $cod_aa{$one}=$thr;     $cod_anti{$one}=$four;      $codnum{$one}=$five;     $tot_cod{$one}=int(1000*$five/$six);      if(!defined($i{$cod_aa{$one}})) { $i{$cod_aa{$one}}=1 }      for($j=$i{$cod_aa{$one}}; $j<=$i{$cod_aa{$one}}+$tot_cod{$one}; $j++) {          $aa_cod{$thr}{$j}=$one;      }      $i{$cod_aa{$one}}=$j;  } }close(GENE);################################################################################################ # #  Read in motifs that are hot or cold to SHM, forassessing output only # #  $hot{ } -> hash containing a list of 4-merhot spots #  $cold{ } -> hash containing a list of 3-mer cold spots ################################################################################################# open(SHM, .”/hotncold.txt”); while (<SHM>) {  if(/{circumflex over ( )}(\S+)\s+(\S+)/) {    $one=$1; $two=$2;    if($one eq ‘COLD’) {        $cold{$two}++;    }    if ($one eq ‘HOT’) {       $hot{$two}++;    }  } } close(SHM);################################################################################################ # #  Read in a library of all 9-mer nucleotide motifs thathave been #  scored for several properties, including # hot SHM motifs,# cold spots, # CpG motifs, #  the length of the longest uninteruptedstretch of the same nucleotide, and a codon usage score # #  $hotsc{ }{} -> hash mapping the tiles 3-mer aa and 9-mer na to the number of SHMhot spots it contains #  $coldsc{ }{ } -> hash mapping the tiles 3-meraa and 9-mer na to the number of SHM cold spots it contains #  $cgsc{ }{} -> hash mapping the tiles 3-mer aa and 9-mer na to the number of CpGmotifs it contains #  $longsc{ }{ } -> hash mapping the tiles 3-mer aaand 9-mer na to the length of its longest stretch of the same na#  $codindexsc{ }{ } -> hash mapping the tiles 3-mer aa and 9-mer na toits aggregate codon usage score ################################################################################################# open(LIB,“gunzip −c ./3mer_library.txt.gz |”); while(<LIB>) {   if (/{circumflex over( )}(\S+)\t(\S+)\t(\d+)\t(\d+)\t(\d+)\t(\d+)\t(\S+)/) {    $hotsc{$1}{$2}=$3;     $coldsc{$1}{$2}=$4;     $cgsc{$1}{$2}=$5;    $longsc{$1}{$2}=$6;     $codindexsc{$1}{$2}=$7;   } } close(LIB);############################################################################################# # # Program begins by reading in a fasta-like file containinga amino acid or nucleic acid sequence # and a second line that containsdesign instructions for each position in the construct # ‘+’ make thisposition hot to SHM # ‘−’ make this position cold to SHM # ‘.’ thisposition is neutral to SHM # # Usage: ./SHMdesign.pl inputfile.fasta A/N#  where either A or N is given to indicate an amino acid or nucleicacid sequence # # $seq -> captures the sequence vector # $change ->captures the design change vector ############################################################################################## open (FILE, “$ARGV[0]”); while (<FILE>) {    if (/\<(\S+)/) {     $change=$1;    }    if (/\>(\S+)/) {      $seq=uc($1);    } }close(FILE);############################################################################################# # # if an amino acid sequence is indicated, a startingnucleic acid sequence is generated that # is consistent with codonusage, and loaded into the arrays listed below. Else, if a nucleic# acid sequence was given as a starting reference the sequence is takendirectly from the # input file and loaded into arrays # # $aa_vector[ ]-> array containing amino acid identities of the sequence # $ch_vector[] -> array containing amino acid identifies of the design changes# $nuc_vector[ ] -> array containing codons for each position # $length-> variable holding the length of the construct in amino acids/codons ############################################################################################## if ($ARGV[1] eq ‘A’) {   @aa_array=split(//,$seq);    foreach $aa (@aa_array) {     chomp $aa;    $count++; $aa_vector[$count]=$aa;   }   @ch_array=split(//,$change);  foreach $ch (@ch_array) {     chomp $ch;    $count2++; $ch_vector[$count2]=$ch;   }   if ($count != $count2) {print “COUNT Mismatch\n”}   for ($length=1; $length<=$count;$length++) {      $r=int(rand(1000)+1);     $nuc_vector[$length]=$aa_cod{$aa_vector[$length]}{$r};   } } elsif($ARGV[1] eq ‘N’) {   $count=0;   @nuc_array=split(//,$seq);     foreach$nuc (@nuc_array) {     chomp $nuc;     $length = int($count/3)+1;$nuc_vector[$length] .= $nuc; $count++;   }   $count2=0;  @ch_array=split(//,$change);     foreach $ch (@ch_array) {     chomp$ch;     $length = int($count2/3)+1; $ch_vector[$length] = $ch;$count2++;   }   if ($count != $count2) {print “COUNT Mismatch\n”}  $templength = int($count/3);   for ($length=1; $length<=$templength;$length++) {     $aa_vector[$length]=$cod_aa{$nuc_vector[$length]};   }} else { print “\n\n input format:\n ./SHMdesign.pl inputfile.fasta A/N\n\n”; exit; }############################################################################ # The program begins the process of construct optimization with 20rounds # of 100 attempted tile substitutions at random positionsthroughout the construct. # At the beginning of each round for ($j=1;$j<=20; $j++) { ############ Print starting state for the round##########################     print “ITERATION\t$j\n”;undef $nuclear;$length2=0; ### Amino acid sequence of construct     for ($i=1;$i<=$length; $i++) {        print “$cod_aa{$nuc_vector[$i]} ”;     }print “\n”; ### Nucleic acid sequence of the construct     for ($i=1;$i<=$length; $i++) {       print “$nuc_vector[$i]”;      @temp=split(//,$nuc_vector[$i]); foreach $n (@temp) { $length2++;      $nuclear[$length2]=$n }     } print “\n”; ### SHM Design vectorfor the construct     for ($i=1; $i<=$length; $i++) {       print“$ch_vector[$i] ”;     }  print “\n”;     for ($i=1; $i<=$length2; $i++){ ### SHM hot spots for the construct    $temp=“$nuclear[$i].”“$nuclear[$i+1].”“$nuclear[$i+2].”“$nuclear[$i+3]”;     if (defined($hot{$temp})) {print “+”} else {print “ ”}     } print“\n”; ### SHM cold spots for the construct     for ($i=1; $i<=$length2;$i++) {      $temp=“$nuclear[$i].”“$nuclear[$i+1].”“$nuclear[$i+2]”;     if (defined($cold{$temp})) { print “-” } else {print “ ”}     }print “\n”; ### CpG motifs within the construct     for ($i=1;$i<=$length2; $i++) {      $temp=“$nuclear[$i].”“$nuclear[$i+1]”;     if ($temp eq ‘CG’) { print “C” } else {print “ ”}     } print “\n”;############# End printing section######################################## ### Substitute 100 3mer aminoacid positions ########################## # # At a randomly chosenposition in the construct, a 9-mer nucleic acid in-frame section ischosen # all other nucleotide sequences consistent with the amino acidsequence are evaluated, # depending on whether this position isdesignated a hot, cold or neutral, and the sequence that results in thebest # design improvement is chosen and subsititued. After a 100interations, the programs evaluates its current state # and prints tothe screen # # $position -> randomly chosen position within theconstruct # $nucleicacid -> current 9-mer nucleic acid at the positionchosen for evaluation # $aminoacid -> current 3-mer amino acid at theposition chosen for evaluation # $better -> flag for best sequencesubstitution at the position, if one is selected # $cur_coldsc,$cur_hotsc, $cur_cgsc, $cur_codindexsc -> place holders for the scores#    of the currently selected 9-mer/3-mer at the position beingevaluated ##########################################################################  for ($k=1; $k<=100; $k++) {     $position=int(rand($length−4))+2;    $pos1=$position−1; $pos2=$position; $pos3=$position+1;    $nucleicacid=“$nuc_vector[$pos1]$nuc_vector[$pos2]$nuc_vector[$pos3]”;$aminoacid=“$cod_aa{$nuc_vector[$pos1]}$cod_aa{$nuc_vector[$pos2]}$cod_aa{$nuc_vector[$pos3]}”;     $cur_hotsc=$hotsc{$aminoacid}{$nucleicacid};    $cur_coldsc=$coldsc{$aminoacid}{$nucleicacid};    $cur_cgsc=$cgsc{$aminoacid}{$nucleicacid};    $cur_longsc=$longsc{$aminoacid}{$nucleicacid};    $cur_codindexsc=$codindexsc{$aminoacid}{$nucleicacid}; #    print“$k\t$position\t$length\t$aminoacid\t$nucleicacid\t#        $cur_hotsc\t$cur_coldsc\t$cur_cgsc\t$cur_longsc\t#        $cur_codindexsc\n”;     undef $better;     if($ch_vector[$pos2] eq ‘−’) {       foreach $spot3 (keys%{$hotsc{$aminoacid}}) {         if (($cur_coldsc <$coldsc{$aminoacid}{$spot3}) &&           ($cur_hotsc >=$hotsc{$aminoacid}{$spot3}) &&           ($cur_cgsc >=$cgsc{$aminoacid}{$spot3}) &&           ($cur_codindexsc <=$codindexsc{$aminoacid}{$spot}) &&           ($longsc{$aminoacid}{$spot}<=4)) {              $better=$spot3;              $cur_coldsc =$coldsc{$aminoacid}{$spot3};              $cur_hotsc =$hotsc{$aminoacid}{$spot3};              $cur_cgsc =$cgsc{$aminoacid}{$spot3};              $cur_codindexsc =$codindexsc{$aminoacid}{$spot};         }       }     }     if($ch_vector[$pos2] eq ‘+’) {       foreach $spot3 (keys%{$hotsc{$aminoacid}}) {         if (($cur_coldsc >=$coldsc{$aminoacid}{$spot3}) &&           ($cur_hotsc <$hotsc{$aminoacid}{$spot3}) &&           ($cur_cgsc >=$cgsc{$aminoacid}{$spot3}) &&           ($cur_codindexsc <=$codindexsc{$aminoacid}{$spot}) &&           ($longsc{$aminoacid}{$spot}<=3)) {              $better=$spot3;              $cur_coldsc =$coldsc{$aminoacid}{$spot3};              $cur_hotsc =$hotsc{$aminoacid}{$spot3};              $cur_cgsc =$cgsc{$aminoacid}{$spot3};              $cur_codindexsc =$codindexsc{$aminoacid}{$spot};         }       }     }     if($ch_vector[$pos2] eq ‘.’) {       foreach $spot3 (keys%{$hotsc{$aminoacid}}) {         if (($cur_cgsc >=$cgsc{$aminoacid}{$spot3}) &&           ($cur_codindexsc <=$codindexsc{$aminoacid}{$spot}) &&           ($longsc{$aminoacid}{$spot}<=3)) {              $better=$spot3;              $cur_coldsc =$coldsc{$aminoacid}{$spot3};              $cur_hotsc =$hotsc{$aminoacid}{$spot3};              $cur_cgsc =$cgsc{$aminoacid}{$spot3};              $cur_codindexsc =$codindexsc{$aminoacid}{$spot};         }       }     }########################################################################################################### # #  if the variable $better is defined afterexhaustively searching for an improved nucleic acid sequence #  at theposition, substitute that sequence into the evolving $nuc_vectorsequence, then proceed with the next trial # #  else, go to the next ofthe 100 random trails and try again ########################################################################################################     if (defined($better)) {      @array=split(//,$better); $tempcount=0; $tempvector[1]=‘’;$tempvector[2]=‘’; $tempvector[3]=‘’;         foreach $nuc (@array) {          chomp $nuc;           $new_position = int($tempcount/3)+1;          $tempvector[$new_position] .= $nuc; $tempcount++;         }#######  print“$nuc_vector[$pos1].$nuc_vector[$pos2].$nuc_vector[$pos3]\t$tempvector[1].$tempvector[2].$tempvector[3]\n”;         $nuc_vector[$pos1]=$tempvector[1];        $nuc_vector[$pos2]=$tempvector[2];        $nuc_vector[$pos3]=$tempvector[3];     }   } } exit;

In addition to the file of potential 3 amino acid tiles shown above, theprogram also calls upon a file of hot spots and cold spots as outlinedbelow, and a listing of the genetic code to translate amino acidsequences to polynucleotide sequences:

TABLE 6 Canonical Hot and Cold Motifs Coldspots Hotspots CCC TACC GGTACTC TACA TGTA GCC TACT AGTA GTC TGCC GGCA GGG TGCA TGCA GAG TGCT AGCAGGC AACC GGTT GAC AACA TGTT AACT AGTT AGCC GGCT AGCA TGCT AGCT AGCT

When a starting amino acid template is given (for instance when theunderlying DNA sequence may not be known), the algorithm begins by firstgenerating a DNA nucleotide sequence that is consistent with both thegiven amino acid sequence and known codon usage in that organism. Thestarting nucleotide template contains an additional line that instructsthe per1 program SHMredesign.p1 as to whether HOT or COLD sites shouldbe incorporated at a given position, making it possible to silence orminimize SHM in portions of evolving proteins, while simultaneouslydirecting SHM to areas for targeting, for instance, the CDRs of anantibody molecule. A given 9-mer in the polynucleotide can be comparedwith all other possible nonameric oligonucleotides that would encode thesame three amino acids at that position.

If a sequence, or portion thereof, is being optimized for SHM (beingmade “hot”), an exhaustive search of all nucleotide sequences consistentwith the amino acid sequence is made, and the nucleotide sequence of theevolving construct is replaced by a new nucleotide sequence if thefollowing conditions are met: (1) the new 9-mer (SHM motif) containsmore hot spots that the existing sequence, (2) the new 9-mer contains anumber of cold spots equal to or less than the evolving sequence, (3)the new 9-mer contains a number of CpG sequence motifs equal to or lessthan the evolving sequence, (4) the evolving sequence has a codon usagescore that equals or improves known aggregate codon usage at theposition, and (5) the sequence does not contain a stretch of any onenucleotide greater than 4 residues.

If a sequence, or portion thereof, is being made resistant to SHM (beingmade “cold”), an exhaustive search of all nucleotide sequencesconsistent with the amino acid sequence is made, and the nucleotidesequence of the evolving construct is replaced by a new nucleotidesequence if the following conditions are met: (1) the new 9-mer (SHMmotif) contains more cold spots that the existing sequence, (2) the new9-mer contains a number of hot spots equal to or less than the evolvingsequence, (3) the new 9-mer contains a number of CpG sequence motifsequal to or less than the evolving sequence, (4) the evolving sequencehas a codon usage score that equals or improves known aggregate codonusage at the position, and (5) the new 9-mer nucleotide sequence doesnot contain a stretch of any one nucleotide greater than 4 residues.

If a sequence is being optimized for other factors other than SHM (beingmade “neutral”), an exhaustive search of all nucleotide sequencesconsistent with the amino acid sequence is made, and the nucleotidesequence of the evolving construct is replaced by the new nucleotidesequence if the following conditions are met: (1) the new 9-mer containsa number of CpG sequence motifs equal to or less than the evolvingsequence, (2) the evolving sequence has a codon usage score that equalsor improves known aggregate codon usage at the position, and (3) the new9-mer nucleotide sequence does not contain a stretch of any onenucleotide greater than 4 residues.

As further described in the priority related application No. 60/902,414,one is able to start from any given polynucleotide sequence and use thisapproach to generate polynucleotide sequences that rapidly converge to asmall number of possible sequences that are optimized for the propertiesdescribed herein.

Following computational analysis, a final optimized polynucleotide canbe synthesized using standard methodology and sequenced to confirmcorrect synthesis. Once the sequence of the polynucleotide has beenconfirmed, the polynucleotide can be inserted into a vector. The vectorcan be introduced into a host cell as described herein and tested forexpression, activity, or increased or decreased susceptibility to SHM.

One of skill in the art will recognize that there are many potentialapproaches, and computational methods which could be used to find thebest codon usage to maximize hot spot or cold spot density, and that theinvention is not intended to be limited to any one specific method ofdetermining the optimum sequence.

As described further below, the creation of synthetic polynucleotidesequences with SHM resistant and or SHM susceptible sequences enablesthe development of novel diversity generating polynucleotide libraries,e.g., seed libraries.

V. Construction of Synthetic Targeted Libraries for SHM MediatedDiversification (“Seed Libraries”)

Static libraries are typically limited in their size and scope. Phagedisplay libraries, for example can display as many as 10¹² members, andribosomal libraries have been constructed that potentially contain ˜10¹⁶members. Libraries presented on the surface of bacterial and mammaliancells are not usually this complex, typically with fewer than 10⁹members. In addition, robust library construction and selection usuallyrequires that libraries contain several fold redundancy, which furtherlimits this theoretically complexity, and makes screening the entirelibrary slow, expensive, and in some cases in-practical.

Despite these levels of complexity, such static libraries can exploreonly a small fraction of possible sequence space, i.e., the potentialnumber of possible permutations within a polynucleotide region ofinterest. For example, a heavy chain IgG sequence may contain more than30 amino acids within the CDR1, CDR2, and CDR3 complementarity regions,giving this single chain more than 20³⁰ possible permutations, dwarfingeven the largest of potential static libraries. Because of thislimitation, researchers have explored methodologies for evolving proteinsequences and libraries. SHM, as addressed in the present application,uses activation induced cytidine deaminase (AID) and error-pronepolymerases as the mechanism for evolving antibody sequences undergoingaffinity maturation. Such a system can facilitate on-going mutagenesisand selection at each position of interest within a polynucleotidelibrary of a given gene and can provide for the selective exploration offunctional sequence space. Such a search strategy enables a much moreproductive region of sequence space to be explored, thereby making themethods described herein very attractive for the rapid development ofnew functionalities and therapeutics.

Additionally, and as discussed below, SHM introduces specific nucleotidetransitions at each position of a “hot spot” motif with a frequency thatcan quantified. This spectrum of nucleotide transitions results indifferent possible silent or non-silent amino acid transitions,depending on which of the three possible reading frames is used. Bydefining the most likely codon transitions mediated by SHM and thesequential flow of mutation events, “preferred hot spot SHM codons” canbe chosen in such a way as to generate a specific panel of amino acidtransitions that can be exploited to enhance the functionality of thelibrary at each amino acid position (see, for example, FIGS. 1, 2, 4 and6).

Thus, the creation of synthetic polynucleotide seed libraries withpreconceived areas of SHM resistant and SHM susceptible regions enablesthe selective directed evolution and selection of proteins, thatmaximally exploits the diversity generating and targeting properties ofSHM.

In the case of antibodies, this typically means targeted diversificationof complementarity determining regions (CDRs) to improve binding to anepitope of interest or to alter the CDRs such that new or alteredepitopes can be bound. Simplified CDR libraries containing four and even2 amino acid alphabets (serine and tyrosine) have also been describedand were found to be capable of binding antigens with high affinity andselectivity. See, e.g., Fellouse F A, Li B, Compaan D M, Peden A A,Hymowitz S G, Sidhu S S Molecular recognition by a binary code. J Mol.Biol. (2005) 348:1153-62; and Fellouse F A, Wiesmann C, Sidhu S SSynthetic antibodies from a four-amino-acid code: a dominant role fortyrosine in antigen recognition. Proc Natl Acad Sci USA. (2004)101:12467-72.

In one aspect, a synthetic gene is one that does naturally undergo SHMwhen expressed in a B cell (i.e., an antibody gene). In another aspect,a synthetic gene is one that does not naturally undergo SHM whenexpressed in a B cell (i.e., a non-antibody gene). In the case ofnon-antibody polypeptides, such as enzymes and other protein classes,this refers to the targeted diversification of regions of the enzyme orprotein of interest which regulates the functional or biologicalactivity of said enzyme or protein, such as, but not limited to, bindingspecificity, enzymatic function, fluorescence, or other properties.Libraries are usually combined with one or more selection strategies asdisclosed below, which allow the improved, or functional members of thelibrary to be separated from the non-functional members of the library.

In one aspect, the present invention includes a synthetic seed librarythat is capable of rapid evolution through AID mediated mutagenesis.This synthetic seed library can have the following properties: i) Thelibrary is easy to synthesize and is based around a limited number ofdiscrete functional sequences; ii) The library contains syntheticpolynucleotide sequences that comprises one or more synthetic variableregions that act as substrates for SHM and include a high density ofpreferred SHM codons, e.g., preferred SHM hot spot codons (see Table 9infra); iii) The library contains synthetic polynucleotide sequencesthat comprises one or more synthetic framework regions that areresistant to SHM mediated mutagenesis and include a low density of SHMhot spots; iv) The library does not contain, or contains a minimumnumber of, certain codons, (“non preferred codons”) that can be mutatedto stop codons in one step through SHM, including, UGG (Trp), UGC (Cys),UCA (Ser), UCG(Ser), CAA(Gln), GAA (Glu) and CAG (Gln); v) From thestarting set of codons, SHM-mediated mutagenesis produces a largepotential diversity at each position selected for mutagenesis, whileminimizing changes through essentional regions of the protein and thecreation of stop codons.

A. Library Design

A library around a specific protein of interest can be designed in lightof any conventional techniques and/or information regarding structureactivity relationships, homology between different species, and x-ray orNMR structural information of the protein, or protein family inquestion. Specific design criteria are provided below, and in relatedapplication No. 60/902,414, entitled “Somatic Hypermutation Systems.”

In certain embodiments of the present invention, initial library designcan involve the following steps:

1. The amino acid sequence of the protein of interest is identified, andthe corresponding polynucleotide sequence determined or reversetranscribed conceptually.

2. Any relevant structural information on the protein of interest, andrelated proteins, or on homologous proteins of interest is obtained.

3. A sequence comparison is preformed on the protein of interestcompared to all other proteins from closely related species, and knownisoforms. In certain embodiments, a sequence alignment would be createdto identify conserved and variable amino acid sequences.

This information can be used to establish whether a specific amino acidor protein region is likely to be important in a functional orstructural, attribute of the protein of interest, and whether it isconserved or variant across functional isoforms of the protein acrossprotein families.

Based on this information, it is possible to establish particularregions of interest that appear to be directly involved in a functionalattribute of the protein of interest. For example, these amino acidswill lie within, or within about 5 Å of a specific functional orstructural attribute of interest. Specific examples include, but are notlimited to, amino acids within CDRs of antibodies, binding pockets ofreceptors, catalytic clefts of enzymes, protein-protein interactiondomains, of co-factors, allosteric binding sites etc.

Based on the structural and sequence analysis as set forth herein, oneor more polynucleotides may be designed to create improved templates forSHM mediated mutagenesis. In certain embodiments, the present inventioncan incorporate one or more of the following concepts:

i) Highly conserved amino acids, or amino acids known, or believed todirectly contribute key binding energy are initially conserved, and thecodon usage within their immediate vicinity changed to either create acold spot motif, or altered to promote mostly conservative amino acidchanges during SHM.

ii) Amino acid domains that appear to be involved in maintaining thecore structural framework of the protein are initially conserved, andtheir codon usage changed to promote mostly conservative amino acidchanges during SHM. Amino acid residues in particularly important framework regions can be altered to use a higher percentage of cold spots,and utilize codons that are resistant, or result in silent mutationsduring SHM.

iii) Amino acids in regions of interest can be varied to incorporatesynthetic variable regions enabling high efficiency SHM, as describedbelow.

iv) Amino acids that are not identified as playing clearly identifiedroles can be codon optimized to enable effective SHM, i.e. the frequencyof SHM hot spots can be maximized and the frequency of SHM cold spotscan be minimized.

B. The Design of Synthetic Variable Regions to Act as Substrates for SHM

The rank ordering of susceptibility to mutagenicity of all SHM hot spotsfor AID and/or error prone polymerases is described above and in SectionIII of priority U.S. Application No. 60/902,414. We further identified areading frame context that is critical for generation of silent vs.non-silent mutations. Herein we describe a synthetic seed libraryapproach that includes the use of a high-density of preferred SHM hotspot codons that can act as a substrate for SHM which can lead to thegeneration of diverse amino acids at each library position which isdesired to be mutated. Such high density SHM motifs are particularlyimportant at the boundary of synthetic variable regions to ensureefficient mutagenesis.

i. WAC Based Motifs

Polynucleotide sequences comprising only the sequence WAC (WAC, whereW=A or T is encoded in equal proportions, and where the reading frame ofreference places C at the wobble or 3^(rd) position of each codon)provides for a high density of hot spots. This pattern produces only 4potential 6-mer nucleotide patterns containing only two codons encodingthe 2 amino acids, Asparagine and tyrosine.

TABLE 7 Codons SEQ ID NO Amino acids AACTAC 298 Asn Tyr AACAAC 299 AsnAsn TACTAC 300 Tyr Tyr TACAAC 301 Tyr Asn

All of the motifs encoded by the WAC library, given in any of the threepossible reading frames, produce a concatenation of hot spots. FIG. 3,which compares these motifs with all other possible 4096 6-mernucleotide combinations for their ability to recruit SHM-mediatedmachinery. Longer assemblies result in the same high density of SHM “hotspots” with no “cold spots.” It is also worth noting that this assemblyof degenerate codons (WACW) results in a subset of possible 4-mer hotspots described by Rogozin et al. (WRCH), where R=A or G, H=A or C or T,and W=T or A.

As seen in FIG. 4, the preferred SHM hot spot codons AAC and TAC, whichcan be the basis for a synthetic library as described herein, can resultin a set of primary and secondary mutation events that createconsiderable amino acid diversity, as judged by equivalent SHM mutationevents observed in Ig heavy chains antibodies. From these two codons,basic amino acids (histidine, lysine, arginine), an acidic amino acid(aspartate), hydrophilic amino acids (serine, threonine, asparagine,tyrosine), hydrophobic amino acids (alanine, and phenylalanine), andglycine are generated as a result of SHM events.

ii WRC Based Motifs

A second potential synthetic high density SHM motif, termed here the WRCmotif (WRC, where W=A or T, R=G or A, C=Cytidine, and where the readingframe of reference places C at the wobble or 3^(rd) position of eachcodon) would be one that contains two possible codons: AGC and TAC.Again four possible 6-mer nucleotides are possible:

TABLE 8 Codons SEQ ID NO Amino acids AGCTAC 294 Ser Tyr AGCAGC 295 SerSer TACAGC 296 Tyr Ser TACTAC 297 Tyr Tyr

The distribution of all 4096 6-mer nucleotide z-scores describing thehotness or coldness of the motif to SHM-mediated mutation is illustratedin FIG. 5. The z-scores for all permuations of 6-mers in the WRCsynthetic library are superimposed on this distrubtion, with the dashedline denoting the top 5% of all possible motifs.

The series of mutation events that lead to the creation of amino aciddiversity, starting from “preferred SHM hot spot codons” AGC and TAC, asobserved in affinity matured IGV heavy chain sequences is illustrated inFIG. 6. 4200 primary and secondary mutation events, starting from codonsencoding asparagine and tyrosine, lead to a set of functionally diverseamino acids.

Again, this motif results in an unusually high density of optimal SHMhot spots and hot codons, as visualized in FIG. 5, when compared withall other 6-mer nucleotide motifs. Like the WAC synthetic motif, the WRCsynthetic motif presents preferred SHM hot spot codons that, whencombined with the SHM activity of AID and one or more error-pronepolymerases, generates a broad spectrum of potential amino aciddiversity at each position (FIG. 6).

Thus, in one aspect, such synthetic preferred SHM motifs (e.g, WAC-basedmotifs and WRC-base motifs) can be targeted to specific regions ofinterest within a polynucleotide sequence that encode specific domains,or sub domains of interest, e.g. a nucleic acid sequence which encodes afunctional portion of a protein, to act as a substrate for SHM and forwhich a high degree of diversity is desired.

In another aspect, preferred SHM motifs (e.g., WAC or WRC motifs) can beinserted systematically throughout the open reading frame of the proteinof interest. For example, for a 100 amino acid residue protein, 300discrete polynucleotides can be generated in which a preferred SHM motif(e.g., WAC or WRC motif) is separately introduced once into everypossible position within the protein. Each of these 100 polynucleotidescan then be screened, either separately, or after being pooled into alibrary, to identify optimal amino acid substitutions at each position.The improved mutations at each position can then be re-combined tocreate a next generation construct comprising all of best individualamino acids identified at each position.

iii. Region Mutagenesis

To provide for effective mutagenesis within larger domains, codons usagecan be modified, as discussed previously to increase the density of hotspots without altering the amino acid sequence, throughout the region ofinterest, e.g, a nucleic acid sequence which encodes a functionalportion of a protein. This approach has the advantage of needing nopreconceived idea of where SHM should be targeted, or what specificamino acids are essential for activity.

In another aspect, for regions in which efficient SHM is required, asynthetic variable region can be created by both changing codon usageand by making conservative amino acid substitutions so as to insertcodons that have an improved hot spot density, to further enhance thedensity of SHM hot spots within a targeted region. Suitable amino acidsubstitutions can be selected from those listed below in Table 9, whileobserving the same overall criteria for stable gene creation, and domainstructure.

TABLE 9 Preferred SHM Codons Use in Codons Amino Acid Group/Sub groupplace of: AGC/AGU Ser Aliphatic/Slightly non polar Thr/Cys GGU GlyAliphatic/Small residue Ala GCU/GCA Ala Aliphatic/Small residue GlyCUA/UUG/CUU Leu Aliphatic/Large Val/Met Charged AAA/AAG LysCharged/Positive Arg CAU His Charged/Positive Arg/Phe GAU AspCharged/Negative Glu GAG Glu Charged/Negative Asp Charged/Polar CAG GlnCharged/Polar Asn AAU/AAC Asn Charged/Polar Gln Aromatic/Phenyl UAU/UACTyr Aromatic/Phenyl Trp UUU/UUA/UUC Phe Aromatic/Phenyl Trp/Phe

In some embodiments, the amino acids Trp, Pro and Gly are conservedwhere their location suggests a functional or structural role. Otherthan these amino acids, if an amino acid to be optimized is not listed,an amino acid from the same sub-group or group as listed above isselected.

In certain embodiments, such synthetic variable regions can beinterspersed with framework regions containing primarily SHM resistantsequences, which can be designed as described previously (see generallyU.S. application No. 60/902,414, entitled “Somatic HypermutationSystems”).

Depending on the amount of information available, a number of distinctlibrary design strategies may be employed, ranging from a veryaggressive targeted approach based on the use of preferred SHM motifs(e.g., WAC or WRC motifs), to a more conservative strategy of usingfairly selective amino acid replacements, to a cautious strategy inwhich only codon usage is changed. An advantage of the present inventionis that each approach results in the generation of only one distinctnucleotide sequence; thus all of these strategies can be subjected toSHM mediated diversity in parallel without significant additionalburden.

C. Sub-Libraries of Improved Variants

Additionally the use of a dynamic evolving system for creating andselecting improved variants of proteins of interest, includingantibodies or binding proteins, as disclosed herein, enables thesequential directed evolution of improved proteins. This can beaccomplished, for example, through the creation of secondary seedlibraries, that comprise SHM optimized sequences at, or around,positions previously identified in the starting, or germline, sequenceto be mutated by AID, and to have direct impact on a specific desiredtrait, for example, in the case of antibodies, improved affinity orcross reactivity. Importantly, such a system enables the on-goingability to analyze the sequences of the variable domains of differentclones to be isolated, and to determine the mutations introduced intothe protein via somatic hypermutation in each case to determine theirdistribution within the clones analyzed; for example, in the case of anantibody, the location of the mutations within the coding region of theheavy and light chains, and their structural context. Mutations soidentified can thus be analyzed based on their position within thestructure of the protein. In certain embodiments, key mutations thatoccur between different evolving clones can be optimized for SHM, andmay then be recombined between, or within families to rapidly generatehybrid antibodies that exhibit favorable increases in affinity orselectivity that represent the sum of all, or a sub set of all mutationsobserved, thereby both maximizing the analysis of useful diversity inthe population, and enabling further evolution of the protein via SHM.Such a conceptual recombination approach enables the rapid evolution ofthe selected antibodies and binding proteins, and avoids the systematicaccumulation of neutral or disadvantageous mutations within thepopulation, and thus provides for significant improvements in bothefficiency and effectiveness in the overall process.

Furthermore, an understanding of the factors that target the somatichypermutation machinery to specific sites within the protein ofinterest, in conjunction with specific insight into how these sequencesare utilized to generate amino acid diversity, enables the developmentof specific algorithms that provide for the predictive creation ofdiversity in a heterologous system undergoing SHM. Such an approach isbased on both an understanding of the amino acids that are likely to becreated, or not created, as a result of SHM acting on a codon, as wellas the temporal sequence of amino acid created that results from SHMacting on a specific, or degenerate codon, or a preferred SHM codon, ora non preferred SHM codon, or any particular SHM motif. This analysisenables the development of DNA constructs that promote or repelmutations in a SHM system, and exhibit efficient and predictablemutagenesis to create diversity in situ.

By combining this understanding with knowledge of the most favorablepositions for mutations actually identified from a highly selectedevolving system, it is possible to create a system that enables therapid and effective mutagenesis of proteins.

As shown in Examples 12 and 13, this approach enables the analysis anddesign of improved seed libraries that has several advantages, includingthe ability to efficiently design low complexity seed libraries that canbe evolved through SHM to create large theoretical complexity which isenriched in functionally improved forms.

Thus in one aspect, the present invention includes a composition ofmatter comprising a seed library of polynucleotides encoding a pluralityof one or more polypeptide species, wherein said polynucleotidescomprise at least one or more codons which have been identified as beingmutated via AID mediated mutagenesis to influence a desired property ofsaid one or more polypeptides, and all, or a subset of all, of said oneor more codons have been altered from the wild type form and optimizedfor somatic hypermutation.

In one non-limiting embodiment of this method, all, or a subset of all,of said one or more codons have been altered from their AID mutated formand optimized for further somatic hypermutation.

The present invention also provides a method of making a protein ofinterest with a desired property, the method comprising the steps of: a.synthesizing a seed library of polynucleotides encoding a plurality ofone or more polypeptide species of interest that have at least oneregion of interest of a protein of interest, wherein the seed library ofpolynucleotides comprise at least one synthetic nucleic acid sequencethat encodes at least one region of interest and has been modified toact as a substrate. for AID mediated somatic hypermutation; b. joiningin operable combination a seed library of polynucleotides encoding aplurality of one or more polypeptide species of interest of a protein ofinterest into an expression vector; c. transforming a host cell with theexpression vector, so that the protein of interest is produced byexpression of the seed library of polynucleotides encoding a plurality.of one or more polypeptide species of interest of a protein of interest;and wherein the host cell expresses AID, or can be induced to expressAID via the addition of an inducing agent; d. optionally inducing AIDactivity, or allowing AID mediated mutagenesis to occur on the seedlibrary; e. identifying a cell or cells within the population of cellswhich expresses a mutated protein having a desired property, and f.establishing one or more clonal populations of cells from the cell orcells identified in step (e).

In other embodiments, provided herein is a method of making a protein ofinterest with a desired or identified property, said method comprisingthe steps of: (a) synthesizing a seed library of polynucleotidesencoding one or more proteins, wherein said seed library ofpolynucleotides comprises at least one synthetic polynucleotide that hasbeen optimized for SHM; (b) joining in operable combination said seedlibrary of polynucleotides into an expression vector; (c) transforming ahost cell with said expression vector, so that said one or more proteinsis produced by expression of said seed library of polynucleotides; andwherein said host cell expresses AID activity or can be induced toexpress AID activity via the addition of an inducing agent; (d) ifneeded, inducing AID activity; (e) identifying a cell or cells withinthe population of cells which express(es) one or more mutated proteinshaving said desired or identified property, and (f) establishing one ormore clonal populations of cells from the cell or cells identified instep (e).

In other embodiments, provided herein is a method of making an antibodyor antigen-binding fragment thereof with a desired property, the methodcomprising the steps of: a. synthesizing a seed library ofpolynucleotides encoding a plurality of one or more antibody heavy chainproteins or fragments that have at least one CDR, wherein thepolynucleotides comprise at least one synthetic nucleic acid sequencethat encodes the at least one CDR and has been modified to act as asubstrate for AID mediated somatic hypermutation; b. synthesizing a seedlibrary of polynucleotides encoding a plurality of one or more antibodylight chain proteins or fragments that have at least one CDR, whereinthe seed library of polynucleotides comprise at least one syntheticnucleic acid sequence that encodes the at least one CDR and has beenmodified to act as a substrate for AID mediated somatic hypermutation;c. joining in operable combination the seed library of polynucleotidesencoding the plurality of antibody heavy chain proteins or fragmentsthereof and the seed library of polynucleotides encoding the pluralityof antibody light chain proteins or fragments thereof into expressionvectors; d. transforming a host cell with the expression vectors, sothat an antibody or an antigen-binding fragment thereof is produced bycoexpression of a heavy chain sequence from the seed library ofpolynucleotides encoding a plurality of antibody heavy chain proteins orfragments thereof and a light chain sequence from the seed library ofpolynucleotides encoding a plurality of antibody light chain proteins orfragments thereof, either on the same or different expression vectors;and wherein the host cell expresses AID, or can be induced to expressAID via the addition of an inducing agent; e. optionally inducing AIDactivity, or allowing AID mediated mutagenesis to occur on the seedlibraries of polynucleotides; f. identifying a cell or cells within thepopulation of cells which expresses a mutated antibody or anantigen-binding fragment thereof having the desired property, and g.establishing one or more clonal populations of cells from the cell orcells identified in step (f).

In other embodiments, provided herein is a method of making an antibodyor antigen-binding fragment thereof with a desired or identifiedproperty, said method comprising the steps of: (a) synthesizing a firstseed library of first polynucleotides encoding a plurality of one ormore antibody heavy chain proteins or fragments thereof that have atleast one heavy chain CDR, wherein said first seed library ofpolynucleotides comprises at least one first synthetic polynucleotidethat has been optimized for SHM; (b) synthesizing a second seed libraryof second polynucleotides encoding said plurality of one or moreantibody light chain proteins or fragments thereof that have at leastone light chain CDR, wherein said second seed library of polynucleotidescomprises at least one second synthetic polynucleotide that has beenoptimized for SHM; (c) joining in operable combination said first andsecond seed libraries of polynucleotides into expression vectors; (d)transforming a host cell with said expression vectors, so that anantibody or an antigen-binding fragment thereof is produced bycoexpression of a heavy chain sequence from said first seed library ofpolynucleotides and a light chain sequence from said second seed libraryof polynucleotides (either on the same or different expression vectors);and wherein said host cell expresses AID activity or can be induced toexpress AID activity via the addition of an inducing agent; (e) ifneeded, inducing AID activity; (f) identifying a cell or cells withinthe population of cells which expresses one or more mutated antibodiesor antigen-binding fragments thereof having the desired or identifiedproperty, and (g) establishing one or more clonal populations of cellsfrom the cell or cells identified in step (f).

In still other embodiments, provided herein is a method of co-evolving aplurality of proteins, the method comprising the steps of: a.synthesizing a first seed library of polynucleotides encoding aplurality of one or more polypeptide species of interest that have atleast one region of interest of a first protein of interest, wherein theseed library of polynucleotides comprise at least one synthetic nucleicacid sequence that encodes the at least one region of interest and hasbeen modified to act as a substrate for AID mediated somatichypermutation; b. synthesizing a second seed library of polynucleotidesencoding a plurality of one or more polypeptide species of interest thathave at least one region of interest of a second protein of interest,wherein the seed library of polynucleotides comprise at least onesynthetic nucleic acid sequence that encodes the at least one region ofinterest and has been modified to act as a substrate for AID mediatedsomatic hypermutation; c. joining in operable combination the seedlibrary of polynucleotides encoding the plurality of polypeptide speciesof interest of the first protein of interest and the seed library ofpolynucleotides encoding the plurality of polypeptide species ofinterest of the second protein of interest into expression vectors; d.transforming a host cell with the expression vectors, so that the firstand second proteins of interest are produced by coexpression of thefirst and second seed libraries of polynucleotides, either on the sameor different expression vectors; and wherein the host cell expressesAID, or can be induced to express AID via the addition of an inducingagent; e. optionally inducing AID activity, or allowing AID mediatedmutagenesis to occur on the seed libraries of polynucleotides; f.identifying a cell or cells within the population of cells whichexpresses a mutated first or second protein of interest having thedesired property, and g. establishing one or more clonal populations ofcells from the cell or cells identified in step (f).

In one aspect, provided herein is a method of co-evolving a plurality ofproteins, said method comprising the steps of: (a) synthesizing a firstseed library of polynucleotides encoding one or more proteins, whereinsaid first seed library of polynucleotides comprise at least one firstsynthetic polynucleotide that has been optimized for SHM; (b)synthesizing a second seed library of polynucleotides encoding one ormore proteins, wherein said second seed library of polynucleotidescomprise at least one second synthetic polynucleotide that has beenoptimized for SHM; (c) joining in operable combination said first andsecond seed libraries of polynucleotides into expression vectors; (d)transforming a host cell with said expression vectors, so that said oneor more first and second proteins are produced by coexpression of saidfirst and second seed libraries of polynucleotides, either on the sameor different expression vectors; and wherein said host cell expressesAID activity or can be induced to express AID activity via the additionof an inducing agent; (e) if needed, inducing AID activity; (f)identifying a cell or cells within the population of cells whichexpresses one or more mutated proteins having the desired or identifiedproperty, and (g) establishing one or more clonal populations of cellsfrom the cell or cells identified in step (f).

In certain aspects, the methods described herein comprise at least onesynthetic nucleic acid sequence that has been modified to act as asubstrate for AID mediated somatic hypermutation by the insertion ofsomatic hypermutation motifs. In certain embodiments, the at least onesynthetic nucleic acid sequence has been modified to act as a substratefor AID mediated somatic hypermutation by the insertion of one or morepreferred SHM codons. In other embodiments, the at least one syntheticnucleic acid sequence has been modified to act as a substrate for AIDmediated somatic hypermutation by the insertion of one or more WACmotif, WRC motif, or a combination thereof.

In one embodiment of any of these methods, the identified codon may bereplaced with a preferred (canonical) SHM codon or preferred (canonical)hot spot SHM codon which introduces a conservative amino acidsubstitution, compared to either the wild-type or AID modified codon. Inanother embodiment of any of these methods, the identified codon may bereplaced with a preferred SHM codon or preferred hot spot SHM codonwhich introduces a semi-conservative mutation at the amino acid level,compared to either the wild-type or AID modified codon. In anotherembodiment of any of these methods, the identified codon may be replacedwith a preferred SHM codon or preferred hot spot SHM codon whichintroduces a non-conservative mutation at the amino acid level comparedto either the wild-type or AID modified codon. In one embodiment,insertion of one or more preferred SHM codons is by insertion of one ormore amino acids substitutions in said region of interest, said aminoacid substitutions being silent, conservative, semi-conservative,non-conservative or a combination thereof. Modifications topolynucleotides made using the methods described herein can render atleast one polynucleotide sequence susceptible or resistant to SHM.

In another aspect, the present invention includes a composition ofmatter comprising a seed library of polynucleotides encoding a pluralityof one or more polypeptide species, wherein said polynucleotidescomprise at least one or more codons which have been identified as beingmutated via AID mediated mutagenesis to influence a desired property ofsaid one or more polypeptides, and all, or a subset of all, of said oneor more codons have been altered from the wild type form and maderesistant to somatic hypermutation.

In certain aspects of the present invention, provided herein arecompositions of matter comprising a seed library of polynucleotidesencoding one or more proteins, wherein said seed library ofpolynucleotides comprises at least one synthetic polynucleotide that hasbeen optimized for SHM by insertion of one or more preferred SHM codons.In other aspects, at least one synthetic polynucleotide has beenoptimized for SHM by reducing the density of non-preferred codons.Synthetic polynucleotides can be made resistant to SHM or madesusceptible to SHM using the methods described herein.

In one non-limiting of this method, all, or a subset of all, of said oneor more codons have been altered from their AID mutated form and maderesistant to somatic hypermutation.

In another aspect, the present invention includes a composition ofmatter comprising a seed library of polynucleotides encoding a pluralityof one or more polypeptide species, wherein said polynucleotidescomprise at least one or more codons which have been identified as beingmutated via AID mediated mutagenesis to influence a desired property ofsaid one or more polypeptides, and a first subset of said one or morecodons have been altered from the wild type form and optimized forsomatic hypermutation, and a second subset of said one or more codonshave been altered from their AID mutated form and made resistant tosomatic hypermutation.

In another aspect, the present invention includes a composition ofmatter comprising a seed library of polynucleotides encoding a pluralityof one or more polypeptide species, wherein said polynucleotidescomprise at least one or more codons which have been identified as beingmutated via AID mediated mutagenesis to influence a desired property ofsaid one or more polypeptides, and a first subset of said one or morecodons have been altered from the AID mutated form and optimized forsomatic hypermutation, and a second subset of said one or more codonshave been altered from their wild type form and made resistant tosomatic hypermutation.

In one aspect of these methods, or any of the methods disclosed herein,the identified codon may be altered without changing the amino acidwhich it encodes, through the replacement of the identified codon by acodon with a higher, or lower probability of SHM. In one aspect, theidentified codon may be replaced with a preferred SHM codon, orpreferred hot spot SHM codon. In another aspect, if the identified codonis a non preferred codon, it may be replaced with a codon of higher,lower, or similar probability of SHM, provided however that thereplacement codon is not also non-preferred.

Alternatively, in another aspect of these methods, the identified codonmay be altered to change both its susceptibility to SHM and the aminoacid which it encodes. In one aspect the identified codon may bereplaced with a preferred SHM codon or preferred hot spot SHM codon.

In one embodiment of any of these methods, the identified codon may bereplaced with a preferred SHM codon or preferred hot spot SHM codonwhich introduces a conservative amino acid substitution, compared toeither the wild-type or AID modified codon. In another embodiment of anyof these methods, the identified codon may be replaced with a preferredSHM codon or preferred hot spot SHM codon which introduces asemi-conservative mutation at the amino acid level, compared to eitherthe wild-type or AID modified codon. In another embodiment of any ofthese methods, the identified codon may be replaced with a preferred SHMcodon or preferred hot spot SHM codon which introduces anon-conservative mutation at the amino acid level, compared to eitherthe wild-type or AID modified codon.

VI. Proteins of Interest

In general, the term “proteins of interest” relates to proteins, orportions thereof, for which it is desired that the polynucleotideencoding the protein is modified for SMH by AID in order to rapidlycreate, select and identify improved variants of that protein. Suchmodified polynucleotides can be made more susceptible to SHM, therebyinducing amino acid changes when the polynucleotide is subjected to AIDas a result of codon usage, and/or the addition of SHM motifs to act assubstrates for AID-mediated SHM and screened for improved function.

Any protein for which the amino acid, or corresponding nucleotidesequence is known, or available (e.g., can be cloned into a vector ofthe present invention) and a phenotype or function can be improved is acandidate for use in the vectors and SHM systems provided herein.Proteins of interest include, for example, surface proteins,intracellular proteins, membrane proteins and secreted proteins from anynaturally occurring or synthetic source. Exemplary, but non-limitingtypes of proteins for use in the synthetic, semi-synthetic and/or seedlibraries provided herein include an antibody heavy chain or portionthereof, an antibody light chain or portion thereof, an enzyme, areceptor, a structural protein, a co-factor, a polypeptide, a peptide,an intrabody, a selectable marker, a toxin, growth factor, peptidehormone, and any other protein which can be optimized, is intended to beincluded.

Biologically active proteins (molecules) also include molecules capableof modulating the pharmacokinetics and/or pharmacodynamics of otherbiologically active proteins (molecules), for example, lipids andpolymers such as polyamines, polyamides, polyethylene glycol and otherpolyethers. For example, polypeptides are those such as, for example,VEGF, VEGF receptor, Diptheria toxin subunit A, B. pertussis toxin, CCchemokines (e.g., CCL1-CCL28), CXC chemokines (e.g., CXCL1-CXCL16), Cchemokines (e.g., XCL1 and XCL2) and CX3C chemokines (e.g., CX₃CL1),IFN-gamma, IFN-alpha, IFN-beta, TNF-alpha, TNF-beta, IL-1, IL-2, IL-3,IL-4, IL-5, IL-6, IL-7, IL-10, IL-12, IL-13, IL-15, TGF-beta, TGF-alpha,GM-CSF, G-CSF, M-CSF, TPO, EPO, human growth factor, fibroblast growthfactor, nuclear co-factors, Jak and Stat family members, G-proteinsignaling molecules such as chemokine receptors, JNK, Fos-Jun, NF-κB,I-κB, CD40, CD4, CD8, B7, CD28 and CTLA-4.

Additionally, there are a variety of other component nucleotidesequences, such as coding sequences and genetic elements that can makeup the core system that one would, in some embodiments, prefer not tohypermutate to maintain overall system integrity. These componentnucleotide sequences include without limitation, i) selectable markerssuch as neomycin, blasticidin, ampicillin, etc; ii) reporter genes (e.g.fluorescent proteins, epitope tags, reporter enzymes); iii) geneticregulatory signals, e.g. promoters, inducible systems, enhancersequences, IRES sequences, transcription or translational terminators,kozak sequences, splice sites, origin of replication, repressors; iv)enzymes or accessory factors used for high level enhanced SHM, or it'sregulation, or measurement, such as AID, pol eta, transcription factors,and MSH2; v) signal transduction components (kinases, receptors,transcription factors) and vi) domains or sub domains of proteins suchas nuclear localization signals, transmembrane domains, catalyticdomains, protein-protein interaction domains, and other protein familyconserved motifs, domains and sub-domains.

In general, one of ordinary skill in the art, based on the teachingherein, would be readily able to select a protein of interest as asuitable candidate for modification to optimize a polypeptide'ssusceptibility to SHM, and devise a suitable assay to monitor thedesired trait of the protein of interest.

Depending on the nature of the protein of interest, and amount ofinformation available on the protein of interest, a practitioner canfollow any combination of the following strategies prior to mutagenesisto create the starting polynucleotide.

1. No codon optimization: Although it may typically be desirable toenhance the number of hot spots within the polynucleotide sequenceencoding a protein of interest, it should be noted that any wild typeprotein will be expected to undergo a certain amount of SHM, and can beused in the present invention without codon optimization, or anyspecific knowledge of the actual sequence. Additionally certainproteins, for example antibodies, naturally comprise polynucleotidesequences which have evolved suitable codon usage, and do not requirecodon modification.

2. Global hot spot optimization: In some aspects, the number of hotspotsin a polynucleotide encoding a protein can be increased, as describedherein. This approach can be applied to the entire coding region of thegene, thereby rendering the entire protein more a more efficientsubstrate for SHM. As discussed herein, this approach may be preferredif relatively little is known about structure activity relationshipswithin the protein, or between related protein isotypes.

3. Selective hot spot modification: Alternatively, as discussed herein,a polynucleotide sequence encoding the protein of interest can beselectively, and or systematically modified through the targetedreplacement of regions of interest, e.g. a nucleic acid sequence whichencodes a functional portion of a protein, with synthetic variableregions, that provide for a high density of hot spots or preferred SHMmotifs which can act as substrates for SHM and seed maximal diversitythrough SHM at specific loci.

One of ordinary skill in the art would understand, based on theteachings provided herein, that any or all of the above approaches maybe undertaken using the present invention. In certain embodiments of thepresent invention, however, global hot spot modification and selectivehot spot modification, can be used together to generate synthetic,semi-synthetic, and/or seed libraries likely to lead to faster and moreefficient generation of diversity in the polynucleotide sequenceencoding a protein, both within specific regions of interest andthroughout the entire protein.

Following design of the required optimized polynucleotide encoding theprotein of interest, it can be synthesized using standard methodologyand sequenced to confirm correct synthesis. Once the sequence of thepolynucleotide has been confirmed, the polynucleotide can be insertedinto a vector of the present invention, and the vector then introducedinto a host cell as described herein to effect mutagenesis.

Once introduced into a suitable hostcell, cells can be induced toexpress AID, and/or other factors to initiate SHM, thereby inducingon-going sequence diversification of the protein of interest. After anappropriate period of time (e.g., 2-5 cell divisions), the resultinghost cells, including variants of the protein of interest, can bescreened and improved mutants identified and separated for the cellpopulation. This process can be iteratively repeated to selectivelyimprove the properties of the protein of interest.

A cell-surface displayed protein can be created through the creation ofa chimeric molecule of a protein of interest coupled in frame to asuitable transmembrane domain. In the case of mammalian cell expression,for example, a MHC type 1 transmembrane domain such as that from H2kk(including peri-transmembrane domain, transmembrane domain, andcytoplasmic domain; NCBI Gene Accession number AK153419) can be used.Likewise the surface expression of proteins in prokaryotic cells (suchas E. coli and Staphylococcus) insect cells, and yeast is wellestablished in the art. For reviews, see for example Winter, G. et al.,Annu. Rev. Immunol. (1994) 12:433-55; Pluckthun, A., (1991)Bio/Technology 9: 545-551; Gunneriusson et al., (1996) J. Bacteriol 781341-1346; Ghiasi et al., (1991) Virology 185 187-194; Boder andWittrup, (1997) Nat. Biotechnol. 15 553-557; and Mazor et al., (2007)Nat. Biotech. 25(5) 563-565.

Surface displayed antibodies or proteins can be created through thesecretion and then binding (or association) of the secreted protein onthe cell surface. Conjugation of the antibody or protein to the cellmembrane can occur either during protein synthesis or after the proteinhas been secreted from the cell. Conjugation can occur via covalentlinkage, by binding interactions (e.g., mediated by specific bindingmembers) or a combination of covalent and non-covalent linkage.

In yet another aspect, proteins can be coupled to a cell through thecreation of an antibody or binding protein fusion protein comprising afirst specific binding member that specifically binds to a target ofinterest fused to a second binding member specific for display on a cellsurface (e.g., in the case of exploiting the binding of protein A and aFc domain: protein A is expressed on and attached to a cell surface andbinds to, and localizes, a secreted antibody (or a protein of interestexpressed as an Fc fusion protein)).

Transfection of appropriate expression vectors containing thecorresponding polynucleotide sequences into suitable mutator positivecells can be performed using any art recognized or known transfectionprotocol. An exemplary surface expressed library of proteins isdescribed in Examples 4 and 5.

Cells expressing a plurality of antibodies or binding proteins from thetransfections above can, optionally, be characterized to select cellsexpressing specific ranges of surface expression of the protein on thecell surface using conventional assays including, but not limited to,FACS.

Staining of light and heavy chain expression can be accomplished, forexample, by using commercially available fluorescein Isothiocyanate(FITC) or R-Phycoerythrin (R-PE) conjugated rat anti-mouse Ig, kappalight chain, and FITC or R-PE conjugated rat anti-mouse Ig G1monoclonalantibodies (BD Pharmingen). Staining can be performed using themanufacture's suggested protocols, usually via incubation of the testcells in the presence of labeled antibody for 30 minutes on ice.Expression levels of cellular antigen expression can be quantified usingSpherotech rainbow calibration particles (Spherotech, Ill.).

Transfected cell populations exhibiting specific ranges of expressioncan be selected. For example, cells with a surface copy number ofgreater than about 10,000, about 50,000, about 100,000, or about 500,000proteins per cell can be selected, and can then be used for efficientaffinity profiling.

Populations of stably transfected cells can be created via, for example,growth for 2 to 3 weeks in the presence of appropriate selectableagents; the resulting cell library can be frozen and stored as a cellbank. Alternatively, cells can be transiently transfected and usedwithin a few days of transfection.

It may be desirable in some instances to convert a surface displayedprotein into a secreted protein for further characterization. Conversioncan be accomplished through the use of a specific linker that can becleaved by incubation with a selective protease such as factor X,thrombin or any other selective proteolytic agent. It is also possibleto include polynucleotide sequences that enable the genetic manipulationof the encoded protein in the vector (i.e., that allow excision of asurface attachment signal from the protein reading frame). For example,the insertion of one or more unique restriction sites, or cre/loxelements, or other recombination elements that enable the selectiveremoval of an attachment signal and subsequent intracellularaccumulation (or secretion) of the protein of interest at will. Furtherexamples include the insertion of flanking loxP sites around anattachment signal (such as a transmembrane domain) allowing forefficient cell surface expression of a protein of interest. However,upon expression of the cre recombinase in the cell, recombination occursbetween the LoxP sites resulting in the loss of the attachment signal,and thus leading to the secretion of the protein of interest.

Once a polypeptide has been optimized to a determined degree, the cellor population of cells expressing an optimized polypeptide of interestcan be isolated or enriched and the phenotype (function) of theoptimized polypeptide can be assayed using art-recognized assays.

Cells can then be re-grown, SHM re-induced, and re-screened over anumber of cycles to effect iterative improvements in the desiredfunction. At any point, the polynucleotide sequence encoding the proteinof interest can be rescued and/or sequenced to monitor on-goingmutagenesis.

For example, episomal plasmid DNA can be extracted (or amplified byco-expression with SV40 T Antigen (J. Virol. (1988) 62 (10) 3738-3746))and then extracted and amplified by PCR using DNA primers that arespecific for the polynucleotide or interest or flanking regions, usingstandard methodology. Alternatively, total RNA can be isolated fromvarious cell populations that have been isolated by flow cytometry ormagnetic beads; episomal DNA and/or total RNA and can be amplified byRT-PCR using primers that are specific for the polynucleotide orinterest or flanking regions using standard methodology. Clones can besequenced using automated DNA sequences from companies such as AppliedBiosystems (ABI-377 or ABI 3730 DNA sequencers). Sequences can beanalyzed for frequency of nucleotide insertions and deletions comparedto the starting sequence.

A. Antibodies and Fragments Thereof

With respect to antibodies, the present invention provides the abilityto bypass the need for immunization in vivo to select antibodies thatbind to key surface epitopes that are aligned with producing the mostrobust biological effects on target protein function. Additionally,mammalian antibodies intrinsically process optimal codon usage patternsfor targeted SHM, greatly simplifying template design strategies. Forcertain antigens, in vivo immunization leads to epitope selection thatdoes not impact target function, thereby hindering the selection ofpotent and efficacious antibody candidates. In still other embodiments,the present invention can provide for the rapid evolution ofsite-directed antibodies that have potent activity by nature of the roleof that epitope in determining target protein function. This providesthe ability to scan target proteins for optimal epitope position andproduce best in class antibodies drugs for use in the clinic.

As described herein, all naturally occurring germline, affinity matured,synthetic, or semi-synthetic antibodies, as well as fragments thereof,may be used in the present invention. In general, such antibodies can bealtered through SHM to improve one or more of the following functionaltraits: affinity, avidity, selectivity, thermostability, proteolyticstability, solubility, folding, immunotoxicity and expression. Dependingupon the antibody format, antibody libraries can comprise separate heavychain and light chain libraries which can be co-expressed in a hostcell. In certain embodiments, full length antibodies can be secreted,and/or surface displayed at the plasma membrane of the host cell. Instill other embodiments, heavy and light chain libraries can be insertedin to the same expression vector, or different expression vectors toenable simultaneous co-evolution of both antibody chains.

In certain embodiments, full length cDNA libraries of naturallyoccurring antibodies, either human or non-human, can be used andsubjected to on-going selection and SHM-mediated mutagenesis using thepresent systems. In other embodiments, all or a portion of a naturallyoccurring antibody, for example an isolated CDR, may be amplified andthe resulting library inserted to an existing naturally occurring, orsynthetic antibody template to create a focused library. In oneembodiment, a library of naturally occurring CDR3 regions may be createdand inserted in a synthetic antibody or fragment thereof, therebycreating a semi-synthetic antibody library.

In one embodiment, increasing the hotspot density in specific subregions of antibodies or fragments thereof (e.g., F(ab′)₂, Fab′, Fab,Fv, scFv, dsFv, dAb or a single chain binding polypeptide) can result intargeted mutagenesis of that region leading to the evolution andselection of a protein with improved characteristics such as one or moreof increased binding affinity, increased binding avidity and/ordecreased non-specific binding. In another embodiment, the use syntheticantibodies with increased hotspots in the constant region (e.g., Fc) canresult in increased binding affinity for an Fc receptor (FcR), therebymodulating signal cascades. Heavy chains and light chains, or portionsthereof, can be simultaneously modified using the procedures describedherein.

Intrabodies used in the methods provided herein can be modified toimprove or enhance folding of the heavy and/or light chain in thereducing environment of the cytoplasm. Alternatively, or in addition, asFv intrabody can be modified to stabilize frameworks that can foldproperly in the absence of intradomain disulfide bonds. Intrabodies canalso be modified to increase, for example, one or more of the followingcharacteristics: binding affinity, binding avidity, epitopeaccessibility, competition with endogenous proteins for the targetepitope, half-life, target sequestration, post-translationalmodification of the target protein, etc. Because intrabodies act withinthe cell, their activity is more analogous to assay methodologiesrequired for enzyme activity assays, which are discussed below insection B.

1. Polynucleotide Identification and Design

A convenient starting point for the creation and evolution of targetedantibody libraries is the use of semi-synthetic libraries that compriseCDRs that are derived from naturally occurring CDR sequences which arereadily available from any suitable donor cells, and which can beligated to pre-defined synthetic human antibody scaffolds. Additionally,because naturally occurring CDRs have evolved with a high hot spotdensity, they make a logical starting place for the development of seedlibraries. Furthermore, the naturally occurring CDR3 sequence includessignificant additional length diversity that is introduced via theaction of terminal transferase activity and which can be exploited forthe development of focused libraries using, for example, CDR1, CDR2 orCDR3 domains of different lengths.

Such libraries comprise (a) a plurality of representative human variabledomain template polynucleotide fragments selected from each of the λ, κ,and H chain antibody isoforms, (b) a plurality of human CDR3 domains ofthe λ, κ, and H variable domains, and (c) one of a plurality of humanconstant region template fragments selected for each of the λ, κ, and Hisoforms, wherein a fragment from each of the pluralities (a)-(c) isligated to a create full-length light and heavy chain sub libraries,which may be subsequently combined to create a master library.

In other embodiments, the antibody libraries comprise multiplerepresentative human variable domains templates which best representgermline sequences which are the commonly used antecedents of maturerecombined antibodies seen in vivo.

In other embodiments, antibody libraries comprise CDR regions of the λ,κ, and H variable domains which are PCR amplified. In other embodimentsthe CDR regions are synthetic, and in one aspect derived from non humanCDR regions.

The semi-synthetic antibody libraries described herein can furthercomprise human constant region templates for each of the λ, κ, and Hisoforms.

Variable Domain Polynucleotide Fragments

As discussed in Example 4, a limited number of human germlinepolynucleotide sequences contribute to the majority λ, κ, and H antibodygenes actually used to generate mature antibodies. The use of theseoptimized scaffolds enables the selection of optimal variable domainsand constant regions that are most relevant to any specific targetclass, and most similar to human therapeutic antibodies.

Each polynucleotide sequences template variable domain is designed toinclude suitable unique restriction sites for sub-cloning, and ligationof CDRs and constant domains. Polynucleotides can be synthesized usingstandard methodology using commercially available vendors (e.g. DNA 2.0,Menlo Park, Calif.) and are sequenced to confirm correct synthesis. Oncethe sequence of the polynucleotide has been confirmed, thepolynucleotide can be inserted into a suitable cloning vector forassembly of the entire antibody chain. In one embodiment, the templatevariable domains lack the CDR3 region.

Amplification of CDRs

In order to prepare a composition of polynucleotides comprising asubstantial portion of the immunological gene repertoire, a startingsource material having the genes coding for the V_(H) and V_(L)polypeptides is required. Preferably the source will be a heterogeneouspopulation of antibody producing cells, i.e. B lymphocytes (B cells). Incertain embodiments, rearranged B cells such as those found in thecirculation (e.g. peripheral blood monocytes), spleen, tonsils or bonemarrow of a vertebrate can be the starting source material. (RearrangedB cells are those in which immunoglobulin gene translocation, i.e.,rearrangement, has occurred as evidenced by the presence in the cell ofmRNA with the immunoglobulin gene V, D and J region transcriptsadjacently located thereon.)

In certain embodiments, it is desirable to bias the repertoire for apreselected activity, such as by using as a source of nucleic acid cells(source cells) from vertebrates in any one of various stages of age,health and immune response. In one embodiment, a healthy animal can berepeatedly immunized prior to collecting rearranged B cells to obtain arepertoire enriched for genetic material producing a ligand bindingtarget polypeptide of high affinity. In other embodiments, a healthyanimal whose immune system has not been recently challenged is used tocollect rearranged B cells thereby producing a repertoire that is notbiased towards the production of genetic material with a high affinityto a target polypeptide.

It should be noted that the greater the genetic heterogeneity of thepopulation of cells from which the polynucleotides are obtained, thegreater the diversity of the immunological repertoire that will be madeavailable for initial screening according to the method of the presentinvention. Thus, cells from different individuals, particularly thosehaving an immunologically significant age difference, and cells fromindividuals of different strains, gender, races or species can beadvantageously combined to increase the heterogeneity of the initialrepertoire.

In certain embodiments of the present invention, the source cells areobtained from a vertebrate, preferably a mammal, which has beenimmunized or partially immunized with an antigenic ligand (antigen)against which activity is sought, i.e., a preselected antigen. Theimmunization can be carried out conventionally. Antibody titer in theanimal can be monitored to determine the stage of immunization desired,which stage corresponds to the amount of enrichment or biasing of therepertoire desired. Partially immunized animals typically receive onlyone immunization and cells are collected therefrom shortly after aresponse is detected. Fully immunized animals display a peak titer,which is achieved with one or more repeated injections of the antigeninto the host mammal, normally at 2 to 3 week intervals. Usually threeto five days after the last challenge, the spleen is removed and thegenetic repertoire of the ° spleenocytes, about 90% of which arerearranged B cells, is isolated using standard procedures. See, CurrentProtocols in Molecular Biology, Ausubel et al., eds., John Wiley & Sons,NY.

The polynucleotides coding for V_(H) and V_(L) polypeptides can bederived from cells producing IgA, IgD, IgE, IgG or IgM, most preferablyfrom IgM and IgG, producing cells.

Methods for preparing fragments of genomic DNA from which immunoglobulinvariable region genes can be cloned as a diverse population are wellknown in the art. See for example Herrmann et al., Methods In Enzymol.,152:180-183, (1987); Frischauf, Methods In Enzymol., 152:183-190 (1987);Frischauf, Methods In Enzymol., 152:190-199 (1987); and DiLella et al.,Methods In Enzymol., 152:199-212 (1987). (The teachings of thereferences cited herein are hereby incorporated by reference.)

The desired gene repertoire can be isolated from either genomic materialcontaining the gene expressing the variable region or the messenger RNA(mRNA) which represents a transcript of the variable region. Thedifficulty in using the genomic DNA from other than non-rearranged Blymphocytes is in juxtaposing the sequences coding for the variableregion, where the sequences are separated by introns. The DNAfragment(s) containing the proper exons must be isolated, the intronsexcised, and the exons then spliced in the proper order and in theproper orientation. For the most part, this can be difficult, so thatthe alternative technique employing rearranged B cells are the method ofchoice because the C D and J immunoglobulin gene regions havetranslocated to become adjacent, so that the sequence is continuous(free of introns) for the entire variable regions.

Where mRNA is utilized, the cells will be lysed under RNase inhibitingconditions. In one embodiment, the first step is to isolate the totalcellular mRNA by hybridization to an oligo-dT cellulose column. Thepresence of mRNAs coding for the heavy and/or light chain polypeptidescan then be assayed by hybridization with DNA single strands of theappropriate genes. Upon isolation of the mRNA representing thetranscripts of the genetic material encoding the variable regions genesof the starting source material, reverse transcription may be performedin a single step or in an optional combined reverse transcription/PCRprocedure to generate a population of cDNA polynucleotidesrepresentative of CDR3 diversity within the starting source material.

In certain embodiments, the present invention includes methods for thegeneration of semi-synthetic antibody libraries that comprise (a) aplurality of variable domain polynucleotide fragments selected from eachof the λ, κ, and H chain antibody isoforms, (b) a plurality of CDR3domains of the λ, κ, and H variable domains, and (c) one of a pluralityof constant region template fragments selected for each of the λ, κ, andH isoforms, wherein a fragment from each of the pluralities (a)-(c) isligated to a create full-length light and heavy chain library. In oneembodiment, the semi-synthetic antibody library is specific for the λisoform. In another embodiment, the semi-synthetic antibody library isspecific for the κ isoform. In still another embodiment, thesemi-synthetic antibody library is specific for the H isoform.

In other embodiments, the semi-synthetic antibody libraries comprisemultiple representative variable domains templates from each of the λ,κ, and H isoforms selected for PCR amplification and/or chemicalsynthesis. These templates are chosen so that each of the λ, κ, and Hisoforms is represented by several variable domains which best representgermline sequence space and which are the commonly used antecedents ofmature recombined antibodies seen in the sequence and structuraldatabases.

In certain embodiments, the representative variable domains can be the λisoform. In certain other embodiments, the representative variabledomains can be the κ isoform. In still other embodiments, therepresentative variable domains can be the H isoform.

In one embodiment of the present invention, the variable domaintemplates are generated by PCR amplification. In another embodiment, thevariable domain templates are generated by chemical synthesis.

In still other embodiments, semi-synthetic antibody libraries compriseCDR3 regions of the λ, κ, and H variable domains which are PCR amplifiedusing primers specific for the 3rd framework region and the constantregion. In one embodiment, the primers are specific for CDR3 regions ofthe λ isoform. In another embodiment, the primers are specific for CDR3regions of the κ isoform. In still another embodiment, the primers arespecific for CDR3 regions of the H isoform.

The semi-synthetic antibody libraries described herein can furthercomprise constant region templates for each of the λ, κ, and H isoformsselected for PCR amplification and/or chemical synthesis. In certainembodiments, the constant region can be the λ isoform. In certain otherembodiments, the constant region can be the κ isoform. In still otherembodiments, the constant region can be the H isoform. In one embodimentof the present invention, the constant region templates are generated byPCR amplification. In another embodiment, the constant region templatesare generated by chemical synthesis.

In other embodiments of the present invention, antibodies can be madeusing synthetic, rather than naturally occurring CDR sequences. Thisapproach enables more rational design strategies to be employed, forexample to enable the development of focused libraries to specificclasses of antigens.

In certain embodiments, to produce the polynucleotides encoding the CDR3regions of the V_(H) chain and V_(L) chain by primer extension, thenucleotide sequence of a primer is selected to hybridize with aplurality of immunoglobulin heavy chain genes at a site substantiallyadjacent to the CDR3 coding region. To hybridize to a plurality ofdifferent CDR3 nucleic acid strands, the primer must be a substantialcomplement of a nucleotide sequence conserved among the differentstrands. In a preferred embodiment, primers are constructed that includeor introduce restriction sites that can then be used to anneal thelibrary with the 3′ end of the template variable regions and theselected constant domains.

If the polynucleotides encoding the CDR3 regions of the V_(H) chain andV_(L) chain are to be produced by polymerase chain reaction (PCR)amplification, two primers must be used for each coding strand ofnucleic acid to be amplified. The first primer becomes part of the sense(plus or coding) strand and hybridizes to a nucleotide sequenceconserved among the polynucleotides which are upstream or span a portionthe CDR3 regions of the V_(H) chain and V_(L) chain within therepertoire. To produce the polynucleotides encoding the CDR3 regions ofthe V_(H) chain, first primers are therefore chosen to hybridize to(i.e. be complementary to) conserved regions within the FR3 region ofimmunoglobulin H isoform genes and the like. Likewise, to produce thepolynucleotides encoding the CDR3 regions of the V_(L)λ and V_(L)κchains, first primers are chosen to hybridize with (i.e. becomplementary to) a conserved region within the FR3 region or which spanthe 5′ portion of the V_(L)λ and V_(L)κ isoform CDR3 region.

Second primers become part of the noncoding (minus or complementary)strand and hybridize to a nucleotide sequence conserved among plusstrands. To produce the polynucleotides encoding the CDR3 regions of theV_(H) chain, second primers are therefore chosen to hybridize with aconserved nucleotide sequence at the 5′ end of the C_(H)-codingimmunoglobulin gene. Likewise, to produce the polynucleotides encodingthe CDR3 regions of the V_(L)λ and V_(L)κ chains, second primers aretherefore chosen to hybridize with a conserved nucleotide sequence atthe 5′ end of the C_(L)-coding immunoglobulin genes.

Preparation of the CDR3 Region Libraries

The strategy used for cloning, i.e., substantially reproducing, thepolynucleotides encoding the CDR3 regions of the Ig V_(H) and V_(L)within the isolated repertoire will depend, as is well known in the art,on the type, complexity, and purity of the polynucleotides making up therepertoire.

In certain embodiments, the method comprises the cloning of thepolynucleotides encoding the CDR3 regions of the V_(H) chain and V_(L)chain from a genetic repertoire comprised of polynucleotide codingstrands, such as mRNA and/or the relevant coding region of genomic DNA.

In one embodiment, the genetic repertoire is in the form of doublestranded genomic DNA, which is usually first denatured, typically bymelting, into single strands. The genomic DNA is subjected to a firstprimary extension reaction by treating (contacting) the DNA with a firstpolynucleotide synthesis primer having a pre-selected nucleotidesequence. The first primer is capable of initiating the first primerextension reaction by hybridizing to a nucleotide sequence, preferablyat least about 10 nucleotides in length and more preferably at leastabout 20 nucleotides in length, conserved within the repertoire. Thefirst primer is sometimes referred to herein as the “sense primer”because it hybridizes to the non-coding or anti-sense strand of anucleic acid and, after one round of priming and extension, becomes anintegrated part of the sense (or coding) strand. In addition, the secondprimer is sometimes referred to herein as the “anti-sense primer”because it hybridizes to a coding or sense strand of a nucleic acid and,after one round of priming and extension, becomes an integrated part ofthe anti-sense (or non-coding) strand.

The first primer extension is performed by mixing the first primer,preferably a predetermined amount thereof, with the polynucleotides ofthe repertoire, preferably a predetermined amount thereof, to form afirst primer extension reaction admixture. The admixture is maintainedunder polynucleotide synthesizing conditions for a time period, which istypically predetermined, sufficient for the formation of a first primerextension reaction product, thereby producing a plurality of differentCDR3 regions polynucleotide complements. The complements are thensubjected to a second primer extension reaction by treating them with asecond polynucleotide synthesis primer having a pre-selected nucleotidesequence. The second primer is capable of initiating the second reactionby hybridizing to a nucleotide sequence, preferably at least about 10nucleotides in length and more preferably at least about 20 nucleotidesin length, conserved among a plurality of different V_(H)-coding genecomplements such as those, for example, produced by the first primerextension reaction. This is accomplished by mixing the second primer,preferably a predetermined amount thereof, with the complement nucleicacids, preferably a predetermined amount thereof, to form a secondprimer extension reaction admixture. The admixture is maintained underpolynucleotide synthesizing conditions for a time period, which istypically predetermined, sufficient for the formation of a first primerextension reaction product, thereby producing a gene library containinga plurality of different polynucleotides encoding the CDR3 regions.

A plurality of first primers and/or a plurality of second primers can beused in each amplification, or an individual pair of first and secondprimers can be used. In any case, the products of amplifications usingthe same or different combinations of first and second primers can becombined to increase the diversity of the gene library.

In an alternate embodiment, the method comprises the cloning of thepolynucleotides encoding the CDR3 regions of the V_(H) chain and V_(L)chain from a genetic repertoire comprised of mRNA by subjecting the mRNAto a reverse transcriptase reaction to yield cDNA. Methods for producingsuch cDNA are well known in the art. The cDNA is subjected to a primerextension reaction similar to the above-described second primerextension reaction, i.e., a primer extension reaction using apolynucleotide synthesis primer capable of hybridizing to a nucleotidesequence conserved among a plurality of different V_(H)-coding genecomplements.

The primer extension reaction is performed using any suitable method.Generally it occurs in a buffered aqueous solution, preferably at a pHof 7-9, most preferably about 8. Preferably, a molar excess (for genomicnucleic acid, usually about 10⁶:1 primer:template) of the primer isadmixed to the buffer containing the template strand. A large molarexcess is preferred to improve the efficiency of the process.

The deoxyribonucleotide triphosphates dATP, dCTP, dGTP, and dTTP arealso admixed to the primer extension (polynucleotide synthesis) reactionadmixture in adequate amounts and the resulting solution is heated toabout 90° C.-100° C. for about 1 to 10 minutes, preferably from 0.5 to 4minutes. After this heating period the solution is allowed to cool tothe calculated annealing temperature or sometimes 20 to 6° C. below thecalculated annealing temperature of the oligonucleotide, which ispreferable for primer hybridization. To the cooled mixture is added anappropriate agent for inducing or catalyzing the primer extensionreaction, and the reaction is allowed to occur under conditions known inthe art. The synthesis reaction may occur at from room temperature up toa temperature above which the inducing agent no longer functionsefficiently. Thus, for example, if thermostable DNA polymerase is usedas inducing agent, the temperature is generally no greater than about40° C.

The inducing agent can be any compound or system which will function toaccomplish the synthesis of primer extension products, includingenzymes. Suitable enzymes for this purpose include, for example, E.coli, DNA polymerase I, Klenow fragment of E. coli DNA polymerase I, T4DNA polymerase, other available DNA polymerases, reverse transcriptase,and other enzymes, including heat-stable enzymes, which will facilitatecombination of the nucleotides in the proper manner to form the primerextension products which are complementary to each nucleic acid strand.Generally, the synthesis will be initiated at the 3′ end of each primerand proceed in the 5′ direction along the template strand, untilsynthesis terminates, producing molecules of different lengths. Theremay be inducing agents, however, which initiate synthesis at the 5′ endand proceed in the above direction, using the same process as describedabove.

The newly synthesized strand and its complementary nucleic acid strandform a double-stranded molecule which can be used in the succeedingsteps of the amplification process.

After producing the DNA homologs representative of the plurality ofpolynucleotides encoding the CDR3 regions of the V_(H) chains and V_(L)chains within the immunological repertoire of the starting sourcematerial, the homologs are typically amplified to produce a quantitysufficient for ligation into the appropriate V_(H) and V_(L) syntheticconstructs. Methods of amplification are known in the art and includesubjecting the DNA homologs to a polymerase chain reaction (PCR) priorto ligating them into the appropriate V_(H) and V_(L) syntheticconstructs. In one such embodiment, the first and/or second primerextension reactions used to produce the gene library are the first andsecond primer extension reactions in a polymerase chain reaction.

PCR is normally carried out by cycling, i.e., simultaneously performingin one admixture, the above described first and second primer extensionreactions, each cycle comprising polynucleotide synthesis followed bydenaturation of the double stranded polynucleotides formed. Methods andsystems for amplifying a DNA homolog are described in U.S. Pat. No.4,683,195 and No. 4,683,202, both to Mullis et al.

In preferred embodiments only one pair of first and second primers isused per amplification reaction. The amplification reaction productsobtained from a plurality of different amplifications, each using aplurality of different primer pairs, are then combined.

The DNA homologs representative of the plurality of polynucleotidesencoding the CDR3 regions produced by PCR amplification are typically indouble-stranded form and have contiguous or adjacent to each of theirtermini a nucleotide sequence defining an endonuclease restriction site.Digestion of the DNA homologs representative of the plurality ofpolynucleotides encoding the CDR3 regions having restriction sites at ornear their termini with one or more appropriate endonucleases results inthe production of DNA homologs having cohesive termini of predeterminedspecificity.

Preparation of Synthetic CDRs

As discussed previously, synthetic CDR sequences can be modified toinclude preferred SHM motifs to act as a substrate for efficient,targeted mutation. In one aspect such SHM motifs may be based on random,semi-random or designed combinations of “WAC” motifs, or “WRC” motifs.Examples of such motifs include any combination of preferred SHM codonsencoding Ser, Tyr and Asn.

In one embodiment such synthetic CDRs comprise at least one sequenceselected from i) to vi)

i) -X₁X₂X₃X₄X₅- (SEQ ID NO: 62) ii) -X₁X₂X₃X₄X₅X₆- (SEQ ID NO: 63) iii)-X₁X₂X₃X₄X₅X₆X₇- (SEQ ID NO: 64) iv) -X₁X₂X₃X₄X₅X₆X₇X₈- (SEQ ID NO: 65)v) -X₁X₂X₃X₄X₅X₆X₇X₈X₉- (SEQ ID NO: 66) vi) -X₁X₂X₃X₄X₅X₆X₇X₈X₉X₁₀- (SEQID NO: 67)

where X₁,X₂,X₃,X₄,X₅,X₆,X₇,X₈,X₉ and X₁₀ are each independently selectedfrom the amino acids Ser, Tyr and Asn.

In one aspect, any one of such motifs may also be separated by one ormore polynucleotide sequences that encode amino acid residues ofparticular interest. In aspect such amino acids are independentlyselected from the group consisting of Gly, Pro, Trp, His, and Met.

In another aspect, such synthetic CDRs may be comprised of random, semirandom or designed, repeating, or non repeating, sequences of Ser, Asnand Tyr up to about 50 amino acids.

In another aspect such synthetic CDRs may be comprised from preferredSHM codons. In one embodiment, such codons (i.e. corresponding to X₁,X₂, X₃ etc above) are independently selected from the group consistingof AGC, UAU, UAC, UUU, UUA, UUC, GCU, GCA, AAA, AAG, GAG, CAG, AAU, AAC,CUA, UUG, CUU, AUU, AUA and AUC.

Synthetic CDRs can range in size from about 5 amino acids to about 40amino acids in length. Longer CDRs specifically CDRs of about 25 toabout 60 amino acids are also contemplated. In certain embodiments ofthe present invention, such synthetic CDRs can comprise at least 50%preferred SHM codons, or more preferably, at least 70% preferred SHMcodons, or most preferred at least 80% preferred SHM codons.

In one aspect of the present invention, a seed library of diversesynthetic CDRs can be constructed in which some, or every position inthe CDR is randomly assigned a preferred SHM codon. Typically thediversity of each such synthetic CDR libraries will range from about 3⁵(3 codons each randomly assigned to all 5 positions) to 3¹⁰ (3 codonseach randomly assigned to all 10 positions) to 13⁵ (top 13 codons eachrandomly assigned to all 5 positions) to 13¹⁰ (top 13 codons eachrandomly assigned to all 10 positions).

In another embodiment, one or more of the synthetic CDR1, CDR2 and CDR3regions of the heavy and light chain are derived from a non-humanantibody. In another embodiment, only the CDR3 regions of the heavy andlight chain are derived from the non-human antibody. In one aspect, suchnon-human CDR sequences comprise synthetic polynucleotide sequences thathave been optimized for somatic hypermutation, and comprise preferredSHM codons and/or preferred SHM hot spot codons. Such synthetic CDRsequences, when incorporated into the human libraries of the presentinvention, provide a method of rapidly humanizing non human antibodiesvia SHM mediated mutagenesis and screening, as described below.

For use herein, each synthetic, variable region can also be designed toinclude suitable unique restriction sites for sub-cloning, and ligationof CDRs and constant domains.

Polynucleotides can be synthesized using standard methodology usingcommercially available vendors (e.g. DNA 2.0, Menlo Park, Calif.) andare sequenced to confirm correct synthesis. Once the sequence of thepolynucleotide has been confirmed, the polynucleotide can be insertedinto a suitable cloning vector, as described above, for assembly of theentire antibody heavy or light chain sub libraries as appropriate. Inone embodiment, the synthetic CDRs are inserted to a synthetic variabledomain template lacking consensus CDR regions, and then ligated intosynthetic constant domains, as described herein with regard tosemi-synthetic full length antibody libraries.

Once each of the sub-libraries has been assembled into one or moreexpression vectors suitable for SHM they may be introduced into a hostcell as described herein to effect mutagenesis.

As described below, specific screens to detect and select surfaceexposed or secreted antibodies with improved traits, typically involveseveral rounds of mutation and selection based on the simultaneousselection of multiple parameters, for example, affinity, avidity,selectivity and thermostability in order to evolve the overall bestantibody.

Information from specific types of libraries, for example, librariescomprising antibodies having a binding specificity to different types ofantigen or libraries comprising CDRs of different lengths, can be usedto aid in the design process for subsequent focused libraries.

2. Template Constant Domains

Any mammalian heavy-chain constant domains (Fc) that correspond to thedifferent antibody classes (i.e. IgA, IgD, IgE, IgG, or IgM) orsubclasses (isotypes), e.g., IgG1, IgG2, IgG3, IgG4, IgA, and IgA2 canbe used as a scaffold, depending on the desired functionality of theantibody. Preferred constant domains include human constant domains ofthe IgG class, and in particular IgG1, IgG2, and IgG4 subclasses.Suitable light chain constant domains include kappa and lambda.Preferred light chain constant domains are human.

Each polynucleotide template constant domain is designed to includesuitable unique restriction sites for sub-cloning, and ligation of CDRsand variable domains. Polynucleotides can be synthesized using standardmethodology using commercially available vendors (e.g. DNA 2.0, MenloPark, Calif.) and are sequenced to confirm correct synthesis. Once thesequence of the polynucleotide has been confirmed, the polynucleotidecan be inserted into a suitable cloning vector for assembly of theentire antibody chain.

3. Library Assembly

In certain embodiments, antibody library assembly involves completingthe assembly of one or more sub-libraries by ligation of the variouselements together.

Specifically, the heavy chain library involves the ligation of the IgV_(H) CDR3 Region Libraries into a cloning vector comprising one or moreof a plurality of chemically synthesized polynucleotides encoding aportion of the template heavy chain variable domain (i.e. lacking itsendogenous CDR3 domain), and a chemically synthesized polynucleotideencoding the template heavy chain constant domain to yield asemi-synthetic antibody library representative of the diversity of arearranged full length heavy chain.

κ Light chain library assembly involves the ligation of the Ig V_(L)κCDR3 Region Libraries into a cloning vector comprising one or more of aplurality of chemically synthesized polynucleotides encoding a portionof the template κ variable domain (i.e. lacking its endogenous CDR3domain) and a chemically synthesized polynucleotide encoding the κ lightchain constant domain to yield a semi-synthetic antibody libraryrepresentative of the diversity of a rearranged full length κ lightchain.

λ Light chain library assembly involves the ligation of the Ig V_(L)λCDR3 Region Libraries into a cloning vector comprising one or more of aplurality of chemically synthesized polynucleotides encoding a portionof the template λ variable domain (i.e. lacking its endogenous CDR3domain) and a chemically synthesized polynucleotide encoding the λ lightchain constant domain to yield a semi-synthetic antibody libraryrepresentative of the diversity of a rearranged full length λ lightchain.

Once each of the sub-libraries has been assembled into one or moreexpression vectors, sub-libraries can be introduced into an appropriatehost cell as described herein. In certain embodiments, each of thesub-libraries is assembled into one or more expression vectors suitablefor SHM, after which the one or more expression vectors suitable for SHMcomprising each of the sub-libraries can be introduced into a host cellas described herein to effect mutagenesis.

4. Screening Methodology

Specific screens to detect and select surface exposed or secretedantibodies with improved traits, are well known in the art, and aredescribed in detail below in Section X. In general, such screens willinvolve several rounds of selection based on the simultaneous selectionof multiple parameters, for example, affinity, avidity, selectivity andthermostability in order to evolve the overall best antibody.

Once an antibody or fragment thereof has been optimized using SHM, thephenotype/function of the optimized antibody or fragment thereof can befurther analyzed using art-recognized assays. Assays for antibodies orfragments thereof include, but are not limited to, enzyme-linkedimmunosorbant assays (ELISA), enzyme-linked immunosorbant spot (ELISPOTassay), gel detection and fluorescent detection of mutated IgH chains,Scatchard analysis, BIACOR analysis, western blots, polyacrylamide gel(PAGE) analysis, radioimmunoassays, etc. which can determine bindingaffinity, binding avidity, etc. Such assays are more fully described inSection X below.

Once optimized antibodies have been identified, episomal DNA can beextracted (or amplified by co-expression with SV40 T Antigen (J. Virol.(1988) 62 (10) 3738-3746)) and then extracted and subjected to PCR usingvariable heavy chain (V_(H)) leader region and/or variable light chain(V_(L)) leader region specific sense primers and isotype specificanti-sense primers. Alternatively, total RNA from selected sorted cellpopulations can be isolated subjected to RT-PCR using variable heavychain (V_(H)) leader region and/or variable light chain (V_(L)) leaderregion specific sense primers and isotype specific anti-sense primers.Clones can be sequenced using standard methodologies and the resultingsequences can be analyzed for frequency of nucleotide insertions anddeletions, receptor revision and V gene selection. The resulting datacan be used to populate a database linking specific amino acidsubstitutions with changes in one or more of the desired properties.Such databases can then be used to recombine favorable mutations or todesign next generation polynucleotide library with targeted diversity innewly identified regions of interest, e.g. nucleic acid sequences whichencode a functional portion of a protein.

B. Non-Antibody Proteins of Interest

With respect to non-antibody proteins, the present invention providesthe ability to bypass the need for in vivo introduction of a library ofrandomly modified proteins to rapidly select modified proteins thatproduce the most robust biological effect or exhibit improved desiredproperties/activities. Thus, the present invention allows for the rapidevolution of improved proteins by scanning target proteins for optimalfunctional and/or structural regions and evolving such regions using themethods described herein. This provides the ability to scan targetproteins for optimal functional region(s) and produce best in classprotein drugs for use in the clinic.

1. Enzymes

Enzymes and pro-enzymes present another category of polypeptides whichcan be readily improved, and for which SHM is useful. Of particularinterest is the application of the present invention to the co-evolutionof multiple enzymatic pathways, involving the simultaneous mutation oftwo or more enzymes. In one aspect, the expression of two syntheticlibraries of polynucleotides encoding proteins of interest in which bothsynthetic polynucleotides libraries are located in proximity to apromoter, and expressed and co-evolved in the same cell simultaneously.In one embodiment, the promoter is a bi-directional promoter such as abi-directional CMV promoter. In another embodiment, the two syntheticlibraries of polynucleotides encoding proteins of interest are placed infront of two uni-directional promoters. The two promoters can be thesame promoter or different promoters. The two synthetic libraries ofpolynucleotides encoding proteins of interest can be in the same vectoror on different vectors. Enzymes and enzyme systems of particular noteinclude, for example, enzymes associated with microbiologicalfermentation, metabolic pathway engineering, protein manufacture,bio-remediation, and plant growth and development.

Many high throughput screening approaches to measure, select and evolveenzymes with improved traits, are well known in the art, and areoutlined in Section X. In general, such screens involve several roundsof selection based on the simultaneous selection of multiple parameters,for example, pH stability, Km, Kcat, thermostability, solubility,proteolytic stability, substrate specificity, co-factor dependency, andtendency for hetero or homo dimerization.

a. Polynucleotide Identification and Design

As described previously, the starting point for mutagenesis is typicallyeither a cDNA clone of the gene of interest, or its amino acid orpolynucleotide sequence. A useful starting point for library developmentis to run a sequence comparison search with this starting sequence usingone of several publicly available databases, for example the PDBdatabase, (www.ncbi.nih.gov/genbank). Such databases include virtuallyall known sequence information and include appropriate analysis tools.

Such searches typically generate information on areas of identity anddivergence between related isoforms of the gene of interest and betweenthe same gene in different organisms.

In addition, the creation of cladograms that show the degree ofrelatedness of different polynucleotide sequences for example by usingthe phylip 3.65 ProtMLK program (see Numerical methods for inferringevolutionary trees. Quarterly Review of Biology 57:379-404) which canprovide important insights on the evolution of related sequences to helpdevelop a template polynucleotide, for example by identifying allenzymes within a specific class or family of interest.

Such genes can be simultaneously evolved by co-expressing AID and orother auxiliary enzymes into a host cell comprising such enzymes. In apreferred case, such enzymes have been codon optimized for SHM.

This approach exploits the ability to identify mutations that not onlyconfer an advantage to specific subsystem in question, but alsopositively impact the overall system which is linked to cell growth andviability.

b. Screening Methodology

Many high throughput screening approaches are well known in the art andcan be readily applied to identify and select improved enzymes (seegenerally, Olsen et al., Methods. Mol. Biol. (2003) 230 329-349; Turner,Trends Biotechnol. (2003) 21(11) 474-478; Zhao et al., Curr. Opin.Biotechnol. (2002) 13(2) 104-110; Mastrobattista et al., Chem. Biol.(2005) 12 (12) 1291-300). In general the screening modality used willdepend on the nature of the enzyme and whether the enzyme of interest isintracellular, or extracellular, and further whether it is membraneassociated or freely secreted.

In general, initial screens that provide useful quantitative informationover a wide dynamic window, and which have a high screening capacity arepreferred. Representative screening approaches include, for example,assays based on the altered ability, or speed of growth of improvedcells, and/or based on the sorting of cells using a flow cytometer, thatcan detect the presence of intracellular fluorogenic reaction productsor altered reporter gene expression (Specific protocols for FACS basedoptimization of enzyme activity are reviewed in the followingreferences; Farinas et al., Comb. Chem. High Throughput Screen (2006)9(4) 321-8; Becker et al., Curr. Opin. Biotechnol. (2004) 15(4) 323-9;Daugherty et al., J. Immunol. Methods (2000) 243 (1-2) 211-227.

Once an enzyme or set of enzymes has been optimized using SHM, acomplete biochemical analysis of the optimized enzyme(s) can be furtheranalyzed using art-recognized assays. Additionally as previouslydiscussed, once optimized enzymes have been identified, episomal DNA canbe extracted or amplified by co-expression with SV40 T Antigen (J.Virol. (1988) 62 (10) 3738-3746), then extracted and subjected to PCRusing specific primers. Alternatively, total RNA can be obtained fromselected cell populations and subjected to RT-PCR using specificprimers. Clones can be sequenced using standard methodologies and theresulting sequences can be analyzed for the frequency of nucleotidemutations. The resulting data can be used to populate a database linkingspecific amino acid substitutions with changes in one or more of thedesired properties. Such databases may then be used to recombinefavorable mutations, or to design next generation polynucleotide librarywith targeted diversity in newly identified regions of interest, e.g.nucleic acid sequences which encode a functional portions of a protein.

2. Receptors

Receptors bind ligands and encompass a broad genus of naturallyoccurring and synthetic polypeptides encoding specific binding members,including, but not limited to, cell-bound receptors such as antibodies(B cell receptors), T cell receptors, Fc receptors, G-coupled proteinreceptors, cytokine receptors, carbohydrate receptors, and Avimer basedreceptors.

In general such receptors will be altered through SHM to improve one ormore of the following traits; affinity, avidity, selectivity,thermostability, proteolytic stability, solubility, dimerization,folding, immunotoxicity, coupling to signal transduction cascades andexpression.

a. Polynucleotide Identification and Design

As described previously, the starting point for mutagenesis is typicallyeither a cDNA clone of the gene of interest, or it's amino acid orpolynucleotide sequence. To maximize the effectiveness of SHM it ispreferred (but not essential) that the starting polynucleotide sequenceis modified to maximize the density of hot spots and to reduce thedensity of cold spots. Such methods are disclosed in sections IV and Vof the present specification.

In general, such receptors possess clearly defined regions that can beeither targeted for mutagenesis through the use of SHM optimizedsequences, or conserved during mutagenesis through the use of SHMresistant sequences. Regions typically targeted for mutagenesis includesites of post-translational modification, surface exposed loop domains,positions of variation between species, protein-protein interactiondomains, and binding domains. Regions typically conserved duringmutagenesis include transmembrane domains, invariant amino acidpositions, signal sequences, and intracellular trafficking domains.Alternatively a scanning approach can be used to systematically inserthot spot motifs throughout the reading frame of the receptor ofinterest, as described previously.

b. Screening Methodology

Many high throughput screening approaches are well known in the art andcan be readily applied to identify and select improved receptors. Ingeneral high throughput screening approaches are preferred.Representative screening approaches include, for example, bindingassays, growth assays, reporter gene assays and FACS based assays.

Once an enzyme or set of enzymes has been optimized using SHM, acomplete pharmacological analysis of the optimized receptor can befurther analyzed using art-recognized assays. Additionally as previouslydiscussed, once an optimized receptor has been identified, episomal DNAcan be extracted or amplified by co-expression with SV40 T Antigen (J.Virol. (1988) 62 (10) 3738-3746), then extracted and subjected to PCRusing specific primers. Alternatively, total RNA can be obtained fromselected cell populations and subjected to RT-PCR using specificprimers. Clones can be sequenced using standard methodologies and theresulting sequences can be analyzed for the frequency of nucleotidemutations. The resulting data can be used to populate a database linkingspecific amino acid substitutions with changes in one or more of thedesired properties. Such databases may then be used to recombinefavorable mutations or to design next generation polynucleotide librarywith targeted diversity in newly identified regions of interest, e.g.,nucleic acid sequences which encodes functional portions of a protein.

VII. Methods for Antibody Humanization

As previously stated, monoclonal antibodies represent a distinct classof biotherapeutics with a great deal of promise. However, thedevelopment of monoclonal antibodies for use in human clinical therapiesis often delayed or prevented due to problems associated with theimmunogenicity of monoclonal antibodies which are derived from non-humansources (i.e., murine monoclonal antibodies). Although it is possible tograft the CDRs of the non human antibody into a human scaffold thistypically results in a significant drop in binding affinity, and as aresult, requires extensive site directed mutagenesis in order to createa high affinity humanized antibody with binding characteristics that arecomparable to the starting non human antibody. In light of this problem,provided herein are methods of rapidly humanizing non-human monoclonalantibodies to reduce their immunogenic activity thereby enabling theiruse as human therapeutics.

In certain aspects, the present invention provides a method forhumanizing a non human antibody, comprising the steps of: a)synthesizing a seed library of polynucleotides encoding one or morehuman antibody heavy chain protein scaffolds comprising at least onesynthetic nucleic acid sequence which encodes all or part of at leastone CDR domain derived from the non human antibody heavy chain protein;b) synthesizing a seed library of polynucleotides encoding a pluralityof one or more human antibody light chain protein scaffolds comprisingat least one synthetic nucleic acid sequence which encodes all or partof at least one CDR domain derived from the non human antibody lightchain protein; c) cloning the antibody heavy chain protein scaffolds andantibody light chain protein scaffolds into expression vectors; d)transforming a host cell with the expression vectors, so that anantibody is produced by coexpression of a heavy chain from the antibodyheavy chain protein scaffolds and a light chain from the light chainprotein scaffolds, e) optionally inducing AID activity in the host cell,or allowing AID mediated mutagenesis to occur on the seed libraries; f)identifying a cell or cells within the population of cells whichexpresses a humanized antibody having binding characteristic of said nonhuman antibody, and g) establishing one or more clonal populations ofcells from the cell or cells identified in step (f).

Library construction for antibody humanization uses the same overallmethodology as discussed above for creation of synthetic andsemi-synthetic antibody libraries in Sections V and VI.

A. Template Variable Domains Identification

The identification of polynucleotide sequences for use as variabledomain templates suitable for humanizing a non-human monoclonal antibodyis typically based on the homology of the non human antibody to knownhuman germline variable domain sequences. Specifically it is preferredthat human variable domains are initially selected that exhibit thegreatest degrees of homology to the non human antibody heavy and lightvariable domains.

In one aspect, the top 10 most related heavy chain variable domaintemplates, and the top 10 most related light chain variable domaintemplates are used to create an initial seed library.

In another aspect, the top 5 most related heavy chain variable domaintemplates, and the top 5 most related light chain variable domaintemplates are used to create an initial seed library.

In one aspect, the top 2 most related heavy chain variable domaintemplates, and the top 2 most related light chain variable domaintemplates are used to create an initial seed library.

Each polynucleotide sequence template variable domain is designed toinclude suitable unique restriction sites for sub-cloning, and ligationof CDRs and constant domains. Polynucleotides can be synthesized usingstandard methodology using commercially available vendors (e.g. DNA 2.0,Menlo Park, Calif.) and are sequenced to confirm correct synthesis. Oncethe sequence of the polynucleotide has been confirmed, thepolynucleotide can be inserted into a suitable cloning vector forassembly of the entire antibody chain. In one embodiment, the templatevariable domains lack the CDR regions.

B. Template Constant Domains

Any polynucleotide sequence encoding a human heavy-chain constantdomains (Fc) that correspond to the different antibody classes (i.e.IgA, IgD, IgE, IgG, or IgM) or subclasses (isotypes), e.g., IgG1, IgG2,IgG3, IgG4, IgA, and IgA2 can be used as a scaffold, depending on thedesired functionality of the antibody. Preferred constant domainsinclude human constant domains of the IgG class, and in particular IgG1,IgG2, and IgG4 subclasses. Suitable human light chain constant domainsinclude kappa and lambda.

Each polynucleotide template constant domain is designed to includesuitable unique restriction sites for sub-cloning, and ligation of CDRsand variable domains. Polynucleotides can be synthesized using standardmethodology using commercially available vendors (e.g. DNA 2.0, MenloPark, Calif.) and are sequenced to confirm correct synthesis. Once thesequence of the polynucleotide has been confirmed, the polynucleotidecan be inserted into a suitable cloning vector for assembly of theentire antibody chain.

C. Non-Human Monoclonal Antibody CDR Regions

The CDR regions of any non-human monoclonal antibody is suitable for usein the methods for humanization described herein. In a preferredembodiment, the synthetically produced CDR regions comprise uniquerestriction sites for ligation of the CDR regions into the humanvariable domains and human constant domains described herein.

In certain embodiments, the polynucleotide sequence encoding all, or aportion of a CDR3 region of a characterized non-human monoclonalantibody can be synthetically produced based upon the known amino acidsequence of the CDR3 region of the monoclonal antibody. In a preferredaspect, the CDR3 polynucleotide sequence has been optimized for somatichypermutation. In one aspect the SHM optimized sequence is optimized forSHM by the insertion of somatic hypermutation motifs. In another aspect,the SHM optimized sequence is optimized for SHM by the insertion of oneor more preferred SHM codons. In another aspect, the SHM optimizedsequence is optimized for SHM by the insertion of one or more WAC motif,WRC motif, or one or more combinations thereof.

In other embodiments, the polynucleotides encoding the CDR3 regions ofthe VH chain and VH chain of the non-human monoclonal antibody can beproduced by polymerase chain reaction (PCR) amplification. As is knownto one of skill in the art, two primers must be used for each codingstrand of nucleic acid to be amplified. The first primer becomes part ofthe sense (plus or coding) strand and hybridizes to a nucleotidesequence conserved among the polynucleotides which are upstream or spana portion the CDR3 regions of the VH chain and VL chain within therepertoire. To produce the polynucleotides encoding the CDR3 regions ofthe VH chain, first primers are therefore chosen to hybridize to (i.e.be complementary to) conserved regions within the FR3 region ofimmunoglobulin H isoform genes and the like. Likewise, to produce thepolynucleotides encoding the CDR3 regions of the VL λ and VL κ chains,first primers are chosen to hybridize with (i.e. be complementary to) aconserved region within the FR3 region or which span the 5′ portion ofthe VL λ and VL κ isoform CDR3 region.

Second primers become part of the noncoding (minus or complementary)strand and hybridize to a nucleotide sequence conserved among plusstrands. To produce the polynucleotides encoding the CDR3 regions of theVH chain, second primers are therefore chosen to hybridize with aconserved nucleotide sequence at the 5′ end of the CH-codingimmunoglobulin gene. Likewise, to produce the polynucleotides encodingthe CDR3 regions of the VL λ and VL κ chains, second primers aretherefore chosen to hybridize with a conserved nucleotide sequence atthe 5′ end of the CL-coding immunoglobulin genes.

Irrespective of the methods used to generate the polynucleotide sequenceencoding the CDR3 region of the monoclonal antibody of interest, oncethe polynucleotide sequence encoding the CDR3 region is isolated it canthen be ligated with the polynucleotide sequences encoding the humanvariable domains and human constant domains described herein to yield apolynucleotide sequence encoding a full length humanized monoclonalantibody.

D. Assembly of the Humanized Monoclonal Antibody Library

In one aspect, the methods described herein for humanizing a heavy chainantibody involve the ligation of all of the non human Ig V_(H) CDRdomains (or in one aspect, only the CDR3 domain) into a plurality ofcloning vectors comprising a polynucleotide described herein whichencodes a plurality of human template heavy chain variable domains (i.e.lacking all endogenous CDR domains, or in one aspect just CDR3), and apolynucleotide encoding the human template heavy chain constant domainto yield a humanized full length heavy chain sub library of themonoclonal antibody of interest.

The methods for humanizing a κ Light chain involve the ligation of theIg VL κ CDRs (or in one aspect, just CDR3) into a plurality of cloningvectors comprising a chemically synthesized polynucleotide describedherein which encodes a plurality of human template κ variable domains(i.e. lacking its endogenous CDR domains, or in one aspect just CDR3)and a chemically synthesized polynucleotide encoding the κ light chainconstant domain to yield a humanized full length κ light chain sublibrary of the monoclonal antibody of interest.

The methods for humanizing a λ Light chain involve the ligation of theIg VL λ CDRs (or in one aspect, just CDR3) into a plurality of cloningvectors comprising a chemically synthesized polynucleotide describedherein which encodes a plurality of human template λ variable domains(i.e. lacking its endogenous CDR domains, or in one aspect just CDR3)and a chemically synthesized polynucleotide encoding the λ light chainconstant domain to yield a humanized full length λ light chain sublibrary of the monoclonal antibody of interest.

Once the full length humanized heavy and light (either κ or λ) chainsub-libraries of the monoclonal antibody of interest have been assembledinto one or more expression vectors, they can be introduced into anappropriate host cell as described herein in Section VIII. In certainembodiments, the full length humanized heavy and light (either κ or λ)chain genes of the monoclonal antibody of interest are assembled intoone or more expression vectors suitable for SHM, after which the one ormore expression vectors suitable for SHM comprising each of the fulllength humanized heavy and light (either κ or λ) chain genes of themonoclonal antibody can be introduced into a host cell as describedherein to effect SHM mediated mutagenesis.

E. Screening Methodology

Specific screens to detect and select surface exposed or secretedhumanized antibodies with improved traits, are well known in the art,and are described in detail below in Section X. In general, such screenswill involve several rounds of selection based on the simultaneousselection of multiple parameters, for example, affinity, avidity,selectivity and thermostability in order to evolve the overall besthumanized antibody.

VIII. Systems for the Expression of Polynucleotide Libraries

In vitro expression and hypermutation systems for use herein includecell free systems that enable the transcription, or coupledtranscription and translation of DNA templates and, in certainembodiments, enable the on-going mutagenesis via SHM. In one embodiment,such in vitro translation systems can be used in combination withribosome display to enable the ongoing mutagenesis and selection ofproteins.

In vitro translation systems include for example the classical rabbitreticulocyte system, as well as novel cell free synthesis systems, (J.Biotechnol. (2004) 110 (3) 257-63; Biotechnol Annu. Rev. (2004) 101-30).Systems for ribosome display are described for example in Villemagne etal., J. 1 mm. Meth. 2006 313 (1-2) 140-148).

In certain embodiments, the synthetic libraries, semi-syntheticlibraries and/or seed libraries described herein can utilize phagedisplay technology by exploiting the capability of bacteriophage toexpress and display biologically functional protein molecule on itssurface. Generally, a phage library can be created by inserting thesynthetic or semi-synthetic libraries described above into gene 3 of M13or T7 phage. Each inserted constructed of the synthetic orsemi-synthetic library is expressed at the N-terminal of the gene 3product, a minor coat protein of the phage. As a result, peptidelibraries that contain diverse peptides can be constructed. The phagelibrary can then be affinity screened against immobilized targetmolecule of interest, such as an antigen, and specifically bound phagesare recovered and amplified by infection into Escherichia coli hostcells. Typically, the target molecule of interest such as a receptor(e.g., polypeptide, carbohydrate, glycoprotein, nucleic acid) isimmobilized by covalent linkage to a chromatography resin to enrich forreactive phage by affinity chromatography) and/or labeled for screenplaques or colony lifts. This procedure is called biopanning. Finally,amplified phages can be sequenced for deduction of the specific peptidesequences.

A variety of solid phases have been used successfully for biopanningwith phage display libraries, including plastic ELISA plates or uncoatedcell culture dishes, magnetic particles, glass beads, and beadedagarose. The most convenient and commonly used solid phase is plasticand the most commonly used method for coating is non-covalentadsorption. However, because the adsorption of proteins onto plasticsurfaces is thought to involve hydrophobic interactions, some ligands,particularly highly hydrophilic proteins or low molecular weightcompounds, may bind inefficiently to plastic unless a covalentattachment method is used. The methods used for the preparation of ELISAplates are directly applicable to biopanning, and detailed ligandimmobilization protocols can be found in enzyme immunoassay laboratorymanuals. To enhance binding, proteins that adsorb poorly to plastic canbe partially denatured with a chaotropic agent such as guanidine, urea,or thiocyanate, or with acid or heat. In addition, target lipids orlipoproteins can be adsorbed to plastic in the presence of deoxycholate.The solid phase used for immobilization of the target ligand usuallydepends on the volume of phage lysate screened. For most applications, aplastic 96-well ELISA plate (e.g., Corning, No. 25801) allows up to 10¹⁰phage to be screened in a single well. However, when larger volumes(>0.2 ml) must be screened, uncoated 6 to 24-well plastic cell cultureplates can be used. When screening very large lysate volumes (>2 ml),plastic Petri dishes can be used. Larger volumes may be required in theinitial rounds of biopanning to ensure that a sufficientlyrepresentative sample has been exposed to the target ligand.

Each panning step starts with a mixture of phage, and seeks to selectfrom that mixture phage whose displayed protein binds the targetreceptor. These phage are specifically “captured” by immobilizing thereceptor (in our case, whole cells) on a solid surface; unbound phageare washed away, and the captured phage are eluted (still in infectiveform), yielding a selected subset of the original phage mixture that iscalled an “eluate.” Usually the eluate from the first round of selectionis amplified by infecting the phage into fresh cells, and the amplifiedeluate then used as input to another round of selection. Altogether, twoor three rounds of selection usually suffice to select for a highlyenriched population of good binders-assuming, of course, the initiallibrary contains such binders.

In other embodiments, an in vitro expression system comprises a libraryof synthetic or semi-synthetic polynucleotides that include anexpression cassette for the expression of the plurality of synthetic orsemi-synthetic polynucleotides encoding a gene of interest. In certainembodiments, the synthetic or semi-synthetic gene comprising a sequencehas been optimized for SHM. For ribosome display, the polynucleotideshould lack a stop codon so that it remained attached to the ribosomeafter translation.

To effect transcription and or translation of the gene of interest, thesystem can include purified or semi-purified components for in vitrotranscription and translation, for example via the use of recombinantfactors with purified 70S ribosomes. In an expression system utilizingongoing SHM, the system would further include recombinant, or purifiedAID and or other factors for SHM/DNA repair.

Cell based expression and hypermutation systems include any suitableprokaryotic or eukaryotic expression system. In certain embodiments, thecell-based expression systems are those that can be used to express AID,can be easily and reliably grown, have reasonably fast growth rates,have well characterized expression systems and can be transformed ortransfected easily and efficiently.

A. Prokaryotic Expression Systems

Within these general guidelines, useful microbial hosts include bacteriafrom the genera Bacillus, Escherichia (such as E. coli), Pseudomonas,Streptomyces, Salmonella, Erwinia, Bacillus subtilis, Bacillus brevis,the various strains of Escherichia coli (e.g., HB101, (ATCC NO. 33694)DH5α, DH10, and MC1061 (ATCC NO. 53338)).

B. Eukaryotic Expression Systems

i. Yeast

Many strains of yeast cells known to those skilled in the art are alsoavailable as host cells for the expression of polypeptides includingthose from the genera Hansenula, Kluyveromyces, Pichia, Rhino-sporidium,Saccharomyces, and Schizosaccharomyces, and other fungi. Preferred yeastcells include, for example, Saccharomyces cerivisae and Pichia pastoris.

ii. Insect Cells

Additionally, where desired, insect cell systems can be utilized in themethods of the present invention. Such systems are described, forexample, by Kitts et al., Biotechniques, 14:810-817 (1993); Lucklow,Curr. Opin. Biotechnol., 4:564-572 (1993); and Lucklow et al. (J.Virol., 67:4566-4579 (1993). Preferred insect cells include Sf-9 and HI5(Invitrogen, Carlsbad, Calif.).

iii. Mammalian Expression Systems

A number of suitable mammalian host cells are also known in the art andmany are available from the American Type Culture Collection (ATCC),10801 University Boulevard, Manassas, Va. 20110-2209. Examples include,but are not limited to, mammalian cells, such as Chinese hamster ovarycells (CHO) (ATCC No. CCL61) CHO DHFR-cells (Urlaub et al., Proc. Natl.Acad. Sci. USA, 97:4216-4220 (1980)), human embryonic kidney (HEK) 293or 293T cells (ATCC No. CRL1573), PER.C6′ cells, or 3T3 cells (ATCC No.CCL92). The selection of suitable mammalian host cells and methods fortransformation, culture, amplification, screening and product productionand purification are known in the art. Other suitable mammalian celllines are the monkey COS-1 (ATCC No. CRL1650) and COS-7 cell lines (ATCCNo. CRL1651), and the CV-1 cell line (ATCC No. CCL70). Further exemplarymammalian host cells include primate cell lines and rodent cell lines,including transformed cell lines. Normal diploid cells, cell strainsderived from in vitro culture of primary tissue, as well as primaryexplants, are also suitable. Candidate cells can be genotypicallydeficient in the selection gene, or may contain a dominantly actingselection gene. Other suitable mammalian cell lines include, but are notlimited to, mouse neuroblastoma N2A cells, HeLa, mouse L-929 cells, 3T3lines derived from Swiss, Balb-c or NIH mice, BHK or HaK hamster celllines, which are available from the ATCC. Each of these cell lines isknown by and available for protein expression.

Also of interest are lymphoid, or lymphoid derived cell lines, such as acell line of pre-B lymphocyte origin. Specific examples include withoutlimitation RAMOS(CRL-1596), Daudi (CCL-213), EB-3 (CCL-85), DT40(CRL-2111), 18-81, (Jack et al., PNAS (1988) 85 1581-1585), Raji cells,(CCL-86) and derivatives thereof.

Suitable vectors for the expression of the synthetic libraries,semi-synthetic libraries, or seed libraries described herein can bebased on any known episomal vector integrating vector, including thosedescribed herein, known in the art, or discovered or designed in thefuture. For use in an SHM system, suitable vectors for the expression ofthe synthetic or semi-synthetic libraries described herein can be basedon any of the vectors described priority U.S. Provisional PatentApplication No. 60/902,414, which can be co-transfected into a host cellendogenously expressing AID. In other embodiments useful in an SHMsystem, suitable vectors for the expression of the synthetic libraries,semi-synthetic libraries, or seed libraries described herein can bebased on any of the vectors described priority U.S. Provisional PatentApplication No. 60/902,414, which can be co-transfected into a host cellwith a separate vector containing the nucleic acid sequence of AID.Expression vectors can also include suitable secretion signals ortransmembrane domains to exert the secretion or surface attachment ofthe protein libraries of interest. In some cases, a surface displayedprotein can be converted into a secreted protein so that the secretedproteins can be further characterized. Conversion can be accomplished,for example, through the inclusion and use of a specific cleavablelinker that can be cleaved by incubation of a selective protease such asfactor X, thrombin or any other selective proteolytic agent. It is alsopossible to include polynucleotide sequences that enable the geneticmanipulation of the encoded protein in the vector (i.e., that allowexcision of a surface attachment signal from the protein reading frame).For example, the insertion of one or more unique restriction sites,cre/lox elements, or other recombination elements that enable theselective removal of an attachment signal. Further examples include theinsertion of flanking loxP sites around the attachment signal (e.g., atransmembrane domain) in the expression vector.

A plasmid encoding the cre recombinase protein (open reading framesynthesized by DNA2.0 and inserted into an expression vector) can betransiently transfected or virally transduced into a cell population ofinterest. Action by the expressed cre recombinase protein leads to thein situ removal of the transmembrane domain portion of the coding regionresulting in translation and production of a secreted form of a proteinin the transfected cell population, which can then be used for furtherstudies.

Representative commercially available viral expression vectors include,but are not limited to, the adenovirus-based Per.C6 system availablefrom Crucell, Inc., the lentiviral-based pLP1 from Invitrogen, and theRetroviral Vectors pFB-ERV plus pCFB-EGSH from Stratagene.

An episomal expression vector suitable for the expression of thesynthetic libraries, semi-synthetic libraries or seed librariesdescribed herein is able to replicate in the host cell, and persists asan extrachromosomal episome within the host cell in the presence ofappropriate selective pressure. (See for example, Conese et al., GeneTherapy 11 1735-1742 (2004)). Representative commercially availableepisomal expression vectors include, but are not limited to, episomalplasmids that utilize Epstein Barr Nuclear Antigen 1 (EBNA1) and theEpstein Barr Virus (EBV) origin of replication (oriP), specific examplesinclude the vectors pREP4, pCEP4, pREP7 from Invitrogen. Theamplification of such OriP based vectors can be achieved via the furtherincorporation of an SV40 origin of replication in the vector, and thetransient expression of the SV40 T antigen.

The vectors pcDNA3.1 (Invitrogen) and pBK-CMV (Stratagene) representnon-limiting examples of an episomal vector that uses T-antigen and theSV40 origin of replication in lieu of EBNA 1 and orip.

An integrating expression vector suitable for the expression of thesynthetic, semi-synthetic libraries, or seed libraries described hereincan randomly integrate into the host cell's DNA, or can include arecombination site to enable the specific recombination between theexpression vector and the host cells chromosome. Such integratingexpression vectors can utilize the endogenous expression controlsequences of the host cell's chromosomes to effect expression of thedesired protein. Examples of vectors that integrate in a site specificmanner include, for example, components of the flp-in system fromInvitrogen (e.g., pcDNAam5/FRT), or the cre-lox system, such as can befound in the pExchange-6 Core Vectors from Stratagene. Examples ofvectors that integrate into host cell chromosomes in a random fashioninclude, for example, pcDNA3.1 (when introduced in the absence ofT-antigen) from Invitrogen, pCI or pFN10A (ACT) Flexi® from Promega.

Alternatively, the expression vector can be used to introduce andintegrate a strong promoter or enhancer sequences into a locus in thecell so as to modulate the expression of an endogenous gene of interest(Capecchi M R. Nat Rev Genet. (2005); 6 (6):507-12; Schindehutte et al.,Stem Cells (2005); 23 (1):10⁻⁵). This approach can also be used toinsert an inducible promoter, such as the Tet-On promoter (U.S. Pat.Nos. 5,464,758 and 5,814,618), in to the genomic DNA of the cell so asto provide inducible expression of an endogenous gene of interest. Theactivating construct can also include targeting sequence(s) to enablehomologous or non-homologous recombination of the activating sequenceinto a desired locus specific for the gene of interest (see for example,Garcia-Otin & Guillou, Front Biosci. (2006) 11:1108-36). Alternativelyan inducible recombinase system, such as the Cre-ER system can be usedto activate a transgene in the presence of 4-hydroxytamoxifen. (Indra etal. Nuc. Acid. Res. (1999) 27 (22) 4324-4327; Nuc. Acid. Res. (2000) 28(23) e99; U.S. Pat. No. 7,112,715).

Elements to be included in an expression vector for use in the presentinvention are well known in the art, and any existing vector can bereadily modified for use in the present invention, for example, throughthe insertion or replacement of one or more polynucleotide sequenceswith synthetic polynucleotide sequences as described above.

IX. Somatic Hypermutation Systems

In one aspect, the polynucleotide libraries (e.g., synthetic libraries,semi-synthetic libraries, and/or seed libraries) of the presentinvention are introduced into a somatic hypermutation system asdescribed in priority U.S. Provisional Patent Application No.60/902,414.

This invention provides for a system that enables mutations be directedto specific genes or regions of interest (made “hot” or SHMsusceptible), and be directed away from structural or marker genes thatare functionally required within the cell or episome, to maintainoverall system functionality and/or stability (made “cold” or SHMresistant). Such systems allow for stable maintenance of a mutagenesissystem that provides for high level targeted SHM in a polynucleotidelibrary of interest, while sufficiently preventing non-specificmutagenesis of structural proteins, transcriptional control regions andselectable markers.

In part, such a system is based around the creation of a more stableversion of cytidine deaminase that can provide for high level sustainedSHM. Additionally, the system includes a variety of other componentnucleotide sequences, such as coding sequences and genetic elements thatcan make up the core system that are optimized for somatic hypermutationand maintain overall system integrity. These component nucleotidesequences include without limitation, i) selectable markers such asneomycin, blasticidin, ampicillin, etc; ii) reporter genes (e.g.,fluorescent proteins, epitope tags, reporter enzymes); iii) geneticregulatory signals, e.g., promoters, inducible systems, enhancersequences, IRES sequences, transcription or translational terminators,kozak sequences, splice sites, origin of replication, repressors; iv)enzymes or accessory factors used for high level enhanced SHM, or it'sregulation, or measurement, such as AID, pol eta, transcription factors,and MSH2; v) signal transduction components (kinases, receptors,transcription factors) and vi) domains or sub domains of proteins suchas nuclear localization signals, transmembrane domains, catalyticdomains, protein-protein interaction domains, and other protein familyconserved motifs, domains and sub-domains.

In one aspect, the vectors described herein comprising the synthetic orsemi-synthetic libraries of the present invention can be transfectedinto a host cell that contains endogenous AID. In another aspect, thevectors described herein comprising the synthetic or semi-syntheticlibraries of the present invention can be co-transfected into a hostcell that contains endogenous AID with a separate vector containing thenucleic acid sequence of AID such that AID is over-expressed in thecell. In yet another aspect, the vectors described herein comprising thesynthetic or semi-synthetic libraries of the present invention can bemodified to include the sequence of cold AID for transfection into ahost cell that does, or does not, contain endogenous AID.

In one embodiment, the cold AID is a mutant form of the enzyme whichexhibits increased mutator activity. Mutant forms of AID can contain astrong nuclear import signal (NLS) a mutation that alters the activityof the nuclear export signal or both.

In one aspect, the mutated AID contains a modified nuclear exportsequence made by one or more mutations independently selected atpositions 180 to 198 of AID (SEQ ID NO: 11), which one or more mutationsenhance mutator activity of the modified AID.

In one embodiment, the modified AID protein has a modified nuclearexport sequence containing at least one mutation selected from amongL181A, L183A, L189A, L196A and L198A. In another embodiment, themodified AID protein has a modified nuclear export sequence containingat least two, at least three or at least four mutations selected fromamong L181A, L183A, L189A, L196A and L198A.

In another aspect, the modified AID protein has a modified nuclearexport sequence containing at least one mutation selected from amongD187E, D188E, D191E, T195I and L198A. In another aspect, the modifiedAID protein has a modified nuclear export sequence containing at leasttwo, at least three or at least four mutations selected from D 187E, D188E, D191E, T195I and L198A.

Mutated AID polypeptides can also contain a nuclear localization signalwhich can be N-terminal or C-terminal. In one non-limiting example, amutated AID can contain a strong nuclear localization signal such as,but not limited to PKKKRKV (SEQ ID NO: 439). In another non-limitingexample, the NLS can be a sequence conforming to the motif K-K/R-X-K/R.

Mutated AID polypeptides described herein can contain both a strong NLSand a modified nuclear export sequence.

In one embodiment, the modified nuclear export sequence can include oneor more of the following mutations: L181A, L183A, L189A, L196A andL198A. In another embodiment, the modified nuclear export sequence caninclude one or more of the following mutations: D187E, D188E, D191E,T195I and L198A.

In any of these mutant forms of AID, the gene may SHM resistant, SHMsusceptible, or can include the appropriate optimal codon usage forexpression of the AID in the host cell of choice without regard for SHMsusceptibility. When used in expression system to target SHM to aprotein of interest, the mutant form of AID can be SHM resistant.

In a preferred embodiment, a SHM system comprising the syntheticlibraries, semi-synthetic libraries, or seed libraries described hereincomprises one or more of the: i) a polynucleotide that has been alteredto positively influence the rate of SHM experienced by thatpolynucleotide, and ii) a polynucleotide that has been altered, tonegatively influence the rate of SHM.

Typically such systems will be used with an expression vector withexpression control sequences to enable the expression of one or morepolynucleotides of interest in a mutator cell line. Suitable expressionvectors can be based on any known viral, or non-viral vector or anartificial chromosome. An expression system can include any combinationof different replicons which can be used in sum to create a coordinatedsystem for SHM.

In another aspect, a SHM system comprising the synthetic orsemi-synthetic libraries described herein can further comprise one ormore expression vectors with one or more of the following additionalelements selected from among: i) an inducible system to regulate theexpression of AID, or an AID homolog, ii) one or more Ig enhancers, iii)one or more E-boxes, iv) one or more auxiliary factors for SHM, v) oneor more factors for stable episomal expression, such as EBNA1, EBP2and/or ori-P, vi) one or more selectable marker genes, vii) one or morefactors to enable the selective amplification of the vectors (i.e. SV40ori and means for expressing SV40 T-Antigen) and viii) any combinationthereof.

If an inducible system is used, such as the Tet-controlled system,doxycycline can be added to the medium to induce expression of thepolynucleotide of interest, or AID for a period of time (e.g., 1 hour(hr), 2 hrs, 4 hrs, 6 hrs, 8 hrs, 10 hrs, 15 hrs, 20 hrs, 24 hrs or anyother time) prior to analysis by an appropriate assay. The cells can beallowed to grow for a certain time to provide for on-goingdiversification, for example, for 1-3 cell generations, or in certaincases 3-6 generations, or in some cases 6 to 10 generations, or longer.

Cells can be iteratively grown, assayed and selected as described hereinto selectively enrich those cells that express a polynucleotide ofinterest exhibiting a desired property. Suitable assay and enrichmentstrategies (e.g., fluorescent activated cell sorting (FACS); affinityseparation, enzyme activity, toxicity, receptor binding, growthstimulation, etc.) are described below.

Once a population of cells has been obtained that is of interest, thepolynucleotides of interest can be rescued and the correspondingmutations sequenced and identified. For example, total mRNA, orextrachromosal plasmid DNA can be amplified by co-expression of SV40 Tantigen (J. Virol. (1988) 62 (10) 3738-3746) and/or can be extractedfrom cells and used as a template for polymerase chain reaction (PCR) orreverse transcriptase (RT)-PCR to clone the modified polynucleotideusing appropriate primers. Mutant polynucleotides can be sub-cloned intoa vector and expressed in E. coli. A tag (e.g., His-6 tag) can be addedto the carboxy terminus to facilitate protein purification usingchromatography.

X. Screening and Enrichment Systems

Polypeptides generated by the expression of the synthetic libraries,semi-synthetic libraries, or seed libraries of polynucleotides describedherein can be screened for improved phenotype using a variety ofstandard physiological, pharmacological and biochemical procedures. Suchassays include for example, biochemical assays such as binding assays,fluorescence polarization assays, solubility assays, folding assays,thermostability assays, proteolytic stability assays, and enzymeactivity assays (see generally Glickman et al., J. BiomolecularScreening, 7 No. 1 3-10 (2002); Salazar et al., Methods. Mol. Biol. 23085-97 (2003)), as well as a range of cell based assays including signaltransduction, motility, whole cell binding, flow cytometry andfluorescent activated cell sorting (FACS) based assays. Cells expressingpolypeptide of interest encoded by a synthetic or semi-synthetic libraryas described herein can be enriched any art-recognized assay including,but not limited to, methods of coupling peptides to microparticles.

Many FACS and high throughput screening systems are commerciallyavailable (see, e.g., Zymark Corp., Hopkinton, Mass.; Air TechnicalIndustries, Mentor, Ohio; Beckman Instruments Inc., Fullerton, Calif;Precision Systems, Inc., Natick, Mass.) that enable these assays to berun in a high throughput mode. These systems typically automate entireprocedures, including all sample and reagent pipetting, liquiddispensing timed incubations, and final readings of the microplate indetector(s) appropriate for the assay. These configurable systemsprovide high throughput and rapid start up as well as a high degree offlexibility and customization. The manufacturers of such systems providedetailed protocols for various high throughput systems. Thus, forexample, Zymark Corp. provides technical bulletins describing screeningsystems for detecting the modulation of gene transcription, ligandbinding, and the like.

A. Cell-based Methods to Measure Activities.

1. Signal Transduction Based Assays

Proteins such as, for example, growth factors, enzymes, receptors andantibodies can influence signal transduction within a cell or cellpopulation, and thereby influence transcriptional activity that can bedetected using a reporter gene assay. Such modulators can behavefunctionally as full or partial agonists, full or partial antagonists,or full or partial inverse agonists.

Thus in one assay format, signal transduction assays can be based on theuse of cells comprising a reporter gene whose expression is directly orindirectly regulated by the protein of interest, which can be measuredby a variety of standard procedures.

Reporter plasmids can be constructed using standard molecular biologicaltechniques by placing cDNA encoding for the reporter gene downstreamfrom a suitable minimal promoter (that is, any sequence that supportstranscription initiation in eukaryotic cells) that sits 5′ to the codingsequence of the reporter gene. A minimal promoter can be derived from aviral source such as, for example: SV40 early or late promoters,cytomegalovirus (CMV) immediate early promoters, or Rous Sarcoma Virus(RSV) early promoters; or from eukaryotic cell promoters, for example,beta actin promoter (Ng, Nuc. Acid Res. 17:601-615, 1989; Quitsche etal., J. Biol. Chem. 264:9539-9545, 1989), GADPH promoter (Alexander, M.C. et al., Proc. Nat. Acad. Sci. USA 85:5092-5096, 1988, Ercolani, L. etal., J. Biol. Chem. 263:15335-15341, 1988), TK-1 (thymidine kinase)promoter, HSP (heat shock protein) promoters, or any eukaryotic promotercontaining a TATA box.

A reporter plasmid also typically includes an element 5′ to the minimalpromoter that contains a consensus recognition sequence, usuallyrepeated 2 to 7 times in a concatenate, to the appropriate branch of thesignal transduction pathway for which monitoring is desired. Examplesinclude, but are not limited to: cyclic AMP response elements (CRE,which responds to changes in intracellular cAMP concentrations,available from Stratagene in phagemid vector pCRE-Luc, Cat. No. 219076),serum response elements (SRE, Stratagene phagemid vector pSRE-Luc. Cat.No. 219080), nuclear factor B response elements (NF-kB, Stratagenephagemid vector pNFKB-Luc Cat. No. 219078), activator protein 1 responseelements (AP-1, Stratagene phagemid vector pAP-1-Luc, Cat. No. 219074),serum response factor response elements (Stratagene phagemid vectorpSRF-Luc, Cat. No. 219082), or p53 binding sites.

Numerous reporter gene systems are known in the art and include, forexample, alkaline phosphatase Berger, J., et al. (1988) Gene 661-10;Kain, S. R. (1997) Methods. Mol. Biol. 63 49-60), .beta.-galactosidase(See, U.S. Pat. No. 5,070,012, issued Dec., 3, 1991 to Nolan et al., andBronstein, I., et al., (1989) J. Chemilum. Biolum. 4 99-111),chloramphenicol acetyltransferase (See Gorman et al., Mol Cell Biol.(1982) 2 1044-51), .beta.-glucuronidase, peroxidase, beta-lactamase(U.S. Pat. Nos. 5,741,657 and 5,955,604), catalytic antibodies,luciferases (U.S. Pat. Nos. 5,221,623; 5,683,888; 5,674,713; 5,650,289;5,843,746) and naturally fluorescent proteins (Tsien, R. Y. (1998) Annu.Rev. Biochem. 67 509-44).

Alternatively, intermediate signal transduction events that are proximalto gene regulation can also be observed, such as, by measuringfluorescent signals from reporter molecules that respond tointracellular changes including, but not limited to, fluctuations incalcium concentration due to release from intracellular stores,alterations in membrane potential or pH, increases in inositoltriphosphate (IP₃) or cAMP concentrations, or release of arachidonicacid.

As used herein, agonists refer to modulators that stimulate signaltransduction and can be measured using various combinations of theconstruct elements listed above. As used herein, partial agonists referto modulators able to stimulate signal transduction to a level greaterthan background, but less than 100% as compared to a full agonist. Asuperagonist is able to stimulate signal transduction to greater than100% as compared to a full agonist reference standard.

As used herein, antagonists refer to modulators that have no influenceon signal transduction on their own, but are able to inhibit agonist-(or partial agonist-) induced signaling. As used herein, partialantagonists refer to modulators that have no influence on signaltransduction on their own, but are able to inhibit agonist- (or partialagonist-) induced signaling to an extent that is measurable, but lessthan 100%.

As used herein, inverse agonists refer to modulators that are able toinhibit agonist- (or partial agonist-) induced signaling, and are alsoable to inhibit signal transduction when added alone.

2. Motility Assays

Agonistic activity on several categories of cell surface molecules(e.g., GPCR's such as chemokine receptors, histamine H4, cannabinoidreceptors, etc.) can lead to cell movements. Thus, partial or fullagonist or antagonist activities of test molecules can be monitored viaeffects on cell motility, such as in chemotaxis assays (Ghosh et al.,(2006) J Med. Chem. May 4; 49(9):2669-2672), chemokinesis (Gillian etal., (2004) ASSAY and Drug Development Technologies. 2(5): 465-472) orhaptotaxis (Hintermann et al., (2005) J. Biol. Chem. 280(9): 8004-8015).

3. Whole Cell Binding Assays

Binding assays that utilize receptors, membrane associated antibodies,and cell surface proteins can be performed using whole cells (as opposedto membrane preparations) in order to monitor activity or bindingselectivity of proteins of interest. Such assays can also be used todirectly select desired cell populations via the use of FACS.(Fitzgerald et al., (1998) J Pharmacol Exp Ther. 1998 November;287(2):448-456; Baker, (2005) Br J. Pharmacol. February; 144(3):317-22)

A large number of fluorescently tagged compounds are available toperform whole cell binding assays. In addition, specific peptides can bereadily labeled in order to profile the binding affinity and selectivityof membrane associated antibodies. In general peptides can be conjugatedto a wide variety of fluorescent dyes, quenchers and haptens such asfluorescein, R-phycoerythrin, and biotin. Conjugation can occur eitherduring peptide synthesis or after the peptide has been synthesized andpurified.

Biotin is a small (244 kilodaltons) vitamin that binds with highaffinity to avidin and streptavidin proteins and can be conjugated tomost peptides without altering their biological activities.Biotin-labeled peptides are easily purified from unlabeled peptidesusing immobilized streptavidin and avidin affinity gels, andstreptavidin or avidin-conjugated probes can be used to detectbiotinylated peptides in, for example, ELISA, dot blot or Western blotapplications.

N-hydroxysuccinimide esters of biotin are the most commonly used type ofbiotinylation agent. N-hydroxysuccinimide-activated biotins reactefficiently with primary amino groups in physiological buffers to formstable amide bonds. Peptides have primary amines at the N-terminus andcan also have several primary amines in the side chain of lysineresidues that are available as targets for labeling withN-hydroxysuccinimide-activated biotin reagents. Several differentN-hydroxysuccinimide esters of biotin are available, with varyingproperties and spacer arm length (Pierce, Rockford, Ill.). Thesulfo-N-hydroxysuccinimide ester reagents are water soluble, enablingreactions to be performed in the absence of organic solvents.

Alternatively, peptides can be conjugated with R-Phycoerythrin, a redfluorescent protein. R-Phycoerythrin is a phycobiliprotein isolated frommarine algae. There are several properties that make R-Phycoerythrinideal for labeling peptides, including an absorbance spectra thatincludes a wide range of potential excitation wavelengths, solubility inaqueous buffers and low nonspecific binding. R-Phycoerythrin also has ahigh fluorescence quantum yield (0.82 at 578 nanometers) that istemperature and pH independent over a broad range. Conjugating peptideswith R-Phycoerythrin can be accomplished using art-recognized techniquesdescribed in, for example, Glazer, A N and Stryer L. (1984). Phycofluorprobes. Trends Biochem. Sci. 9:423-7; Kronick, M N and Grossman, P D(1983) Immunoassay techniques with fluorescent phycobiliproteinconjugates. Clin. Chem. 29:1582-6; Lanier, L L and Loken, M R (1984)Human lymphocyte subpopulations identified by using three-colorimmunofluorescence and flow cytometry analysis: Correlation of Leu-2,Leu-3, Leu-7, and Leu-11 cell surface antigen expression. J. Immunol.,132:151-156; Parks, D R et al. (1984) Three-color immunofluorescenceanalysis of mouse B-lymphocyte subpopulations. Cytometry 5:159-68;Hardy, R R et al. (1983) demonstration of B-cell maturation in X-linkedimmunodeficient mice by simultaneous three-color immunofluorescence.Nature 306:270-2; Hardy R R et al. (1984) J. Exp. Med. 159:1169-88; andKronick, M N (1986) The use of phycobiliproteins as fluorescent labelsin immunoassay. J Immuno. Meth. 92:1-13.

A number of cross-linkers can be used to produce phycobiliproteinconjugates including, but not limited to, N-Succinimidyl3-[2-pyridyldithio]-propionamido, (Succinimidyl6-(3-[2-pyridyldithio]-propionamido)hexanoate, or (Sulfosuccinimidyl6-(3-[pyridyldithio]-propianamido)hexanoate. Such cross-linkers reactwith surface-exposed primary amines of the phycobiliprotein and createpyridyldisulfide group(s) that can be reacted with peptides that containeither free sulfhydryl groups or primary amines.

Another option is to label peptides with fluorescein isothiocyanate(molecular weight 389). The isothiocyanate group on the fluorescein willcross-link with amino, sulfuhydryl, imidazoyl, tyrosyl or carbonylgroups on peptides, but generally only derivatives of primary andsecondary amines yield stable products. Fluorescein isothiocyanate hasan excitation and emission wavelengths at 494 and 520 nanometersrespectively and a molar extinction coefficient of 72,0000 M⁻¹ cm⁻¹ inan aqueous buffer at pH 8 (Der-Balian G, Kameda, N. and Rowley, G.(1988) Fluorescein labeling of Fab while preserving single thiol. Anal.Biochem. 173:59-63).

4. Whole Cell Activity Assays

Many proteins, including enzymes, Intrabodies and receptors can bedirectly assayed within a living cell, or when surface displayed on thesurface. Typically for successful FACS based screening a fluorescent orfluorogenic membrane permeant substrate is required, many such reagentsare commercially available, for example from Molecular Probes(Invitrogen, Calif.). An increase in enzyme activity typically resultsin increased production of a fluorescent product that is trapped withinthe cell resulting in cells with more fluorescence which can beseparated from less fluorescent cells, for example by FACS. Additionallymany high throughput microplate screens exist for screening of proteinlibraries that exploit virtually any existing assay of enzymaticactivity, see generally, Geddie, et al., Meth. Enzymol. 388 134-145(2004).

5. Cell Growth Assays

The expression, or activity of a variety of proteins such as, forexample, growth factors, enzymes, receptors and antibodies can influencethe rate of growth of a host cell which be exploited either as an assay,or as a means of separating improved proteins.

Thus in one assay format, cells can be diluted to a limiting dilutionand cells which grow more rapidly detected and selected. In one aspectsuch growth based assays can involve the ability to grow in the presenceof a new substrate for which an improved enzymatic pathway of metabolismis required, for example a new carbon source. In another embodiment,growth assays can involve selection in the presence of a toxin, where ade-activation mechanism for the toxin is required. In another case,growth can be desired in response to the presence of a specific ligand,where high affinity binding of the ligand is required.

B. Selection and Enrichment Strategies

1. Flow Cytometry and FACS

Flow cytometry and the related flow sorting (also known as fluorescenceactivated cell sorting, or FACS) are methods by which individual cellscan be quantitatively assayed for the presence of a specific componentor component variant based upon staining with a fluorescent reporter.Flow cytometry provides quantitative, real time analysis of livingcells, and can achieve efficient cell sorting rates of 50,000cells/second, and is capable of selecting individual cells or definedpopulations. Many commercial FACS systems are available, for example BDBiosciences (CA), Cytopeia (Seattle, Wash.) Dako Cytomation (Australia).

A FACS can be equipped with a variety of lasers, which can produce awide range of available wavelengths for multiple parameter analysis, andfor use with different fluorophores. Classically the water cooled ionlasers using argon, krypton, or a mix of both can produce severalspecific lines; 408 nm, 568 nm, and 647 nm for example are majoremission lines for Krypton; 488 nm, 457 nm, and others are argon lines.These lasers require high voltage multiphase power and cooling water,but can produce high power outputs. Additionally tunable and non tunablediode lasers exist, for example a 408 nm line can be stably created viaa light emitting diode (LED) and this can be easily added to a sorter.Additionally dye lasers can be used to further extend the range ofavailable wavelengths available for FACS analysis.

During FACS analysis, cells are stained with the specific reporter andthen hydrodynamically focused into a single cell steam for interrogationwith a laser which excites the fluorescent moiety. Fluorescent emissionis detected through a wavelength restricted optical pathway andconverted to numeric data correlated to an individual cell. In the caseof flow sorting, predefined subsets of emission criteria can be met andthe cells of interest diverted into a collection receptacle for furtheruse by electrostatic repulsion or mechanical action (Herzenberg L A,Sweet R G, Herzenberg L A: Fluorescence activated cell sorting, Sci Amer234(3): 108, March 1976).

FACS based approaches are compatible with signal transduction basedassays, activity based assays, and binding assays, and with a widevariety of proteins of interest, including for example, antibodies,receptors, enzymes and any surface displayed protein. FACS can beefficiently applied to most mammalian, yeast and bacterial cells, aswell as fluorescently tagged beads.

In one embodiment, FACS can be used to screen a library of cellsexpressing surface displayed proteins (e.g., surface displayedantibodies) that are undergoing, or have undergone, SHM mediateddiversity. In this approach, a cell surface displayed library is usedand the displayed proteins are first incubated with fluorescently taggedantigen in solution. The FACS instrument is able to separate the highaffinity protein members of the library, which have greater fluorescenceintensity, from the lower affinity members. The use of optimized bindingprotocols in conjunction with FACS based selection has been shown to becapable of evolving antibodies with up to femtomolar affinities, See,e.g., Boder et al. PNAS, (2000) 97: 10701-10705; Boder et al., (2000)Meth. Enzymol. (2000) 328: 430-444; VanAntwerp et al., Biotechnol. Prog.(2000) 16: 31-37).

In order to effectively select and rapidly evolve, the antibodies andbinding proteins which have high affinity to an antigen of interest,protocols can be established that can facilitate the isolation ofantibodies with a broad range of affinities to the antigens of interest,and yet eliminate proteins that bind to labeling or coupling reagents.These protocols involve both a progression in the stringency of the cellpopulation selected, and a decrease in the concentration and density ofthe target antigen presented to the cells.

With respect to the stringency or fraction of the total cell populationcollected during each round of selection, initial screens will generallyuse relatively low discrimination factors in order to capture as manyproteins as possible that possess small incremental improvements inbinding characteristics. For example, a typical initial sort may capturethe top 10%, top 5% or top 2% of all cells that bind a target. Largeimprovements in affinity may be the result of combinations of mutations,each of which contribute small additive effects to overall affinity.(Hawkins et al., (1993) J. Mol. Biol. 234: 958-964). Therefore, recoveryof all library clones with even marginally improved affinities (2-3fold) is desirable during the early stages of library screening, andsorting gates can be optimized to recover as many clones as possiblewith minimum sacrifice in enrichment.

These selected cells can subsequently be allowed to recover and grownusing standard culture conditions for a number of days until thepopulation has reached a reasonable number to allow for a subsequentround of FACS sorting, analysis, mutagenesis, cell banking, or todetermine sequence information. As discussed below, subsequent rounds ofselection to identify higher affinity binders can be achieved byprogressively decreasing the density and concentration of labeledbinding peptide used in the preincubation steps prior to FACS analysis.

Following a successful first round of sorting, the collected cells canbe re-grown to amplify the population and then resorted. At this, andsubsequent stages of sorting, greater enrichments are possible sincemore copies of each desirable clone are present within the examined cellpopulation. For example only about the top 1%, top 0.5%, top 0.2%, ortop 0.1% of the cells in the population may be selected in order toidentify significantly improved clones. With respect to establishingoptimal binding and selection strategies, first generation hits,including germline antibodies, typically have low affinities andrelatively rapid off rates. For example, Sagawa et al. (Mol. Immunology,39: 801-808 (2003)) observed that the apparent affinity for germline Absis typically in the range of 2×10⁴ to 5×10⁶ M⁻¹, but that this affinityincreases to around 10⁹ M⁻¹ during affinity maturation (i.e., an effectthat is mediated primarily by decreasing the off rate (K_(off))).

The binding characteristics of weak binding antibodies may slow thescreening of early generation, non-optimized libraries because specific,but low affinity binding antibodies typically have rapid off rates andtend therefore tend to be lost during wash steps. Loss of these specificbinders may result in the isolation of antibodies that bindnon-specifically to components used in the selection process (Cumbers etal., Nat. Biotechnol. 2002 November; 20(11): 1129-113).

To maximize the selection of proteins with relatively low affinities(i.e., having a Kd greater than about 500 nM), binding interactions arestabilized to prevent the dissociation of binding peptides during thescreening process, and include appropriate blocking reagents toeliminate binding to coupling reagents and support matrices. To achievethis goal, initial screens should use fluorescently tagged beads loadedwith a high density of antigens to exploit avidity effects, based on theuse of multiple binding interactions to increase the binding strength oflow affinity interactions, while also including pre-incubations withcoupling and labeling reagents such as streptavidin, avidin, and nakedbeads etc., to eliminate non-specific binding (see generally, Aggarwalet al., (2006) Bioconjugate Chem. 17 335-340; Wrighton et al., (1996)Science 273 458-64; Terskikh et al. (1997) PNAS 94 1663-8; Cwirla etal., (1997) Science 276 1696-9; and Wang et al. (2004) J. Immunologicalmethods 294 23-35).

By careful control of bead loading density, washing and pre-incubationconditions it has been demonstrated that even such low affinity bindinginteractions can be reproducibly monitored, (Werthen et al., (1993) BBA326-332). Importantly these improvements to binding efficiency have beendemonstrated to occur without any significant increase in non-specificreactivity (Giordano et al., (2001) Nat. Med. 7 1249-53). As discussedabove, selections generally will also be based on using a relatively lowstringency cut off during FACS to ensure that all of these weak bindinglibrary members are selected.

To further eliminate non-specific members of the library (i.e., thosethat bind to the beads, or coupling reagents, rather than the bindingpeptides), the resultant cell populations are screened directly witheither polymeric binding peptide or intact polymeric antigen usingdistinct coupling reagents (e.g., via the use of biotinylated antigencoupled to streptavidin-fluorophore conjugate to form anantigen-streptavidin fluorescent complex). Coupling or labeling of thebinding peptide to biotin or fluorophores can be achieved usingstandard, art-recognized protocols, as described herein and in theExamples.

Streptavidin binds biotin with femtomolar affinity and forms tetramersin physiological conditions, thereby generating a tetravalent complexwhen preincubated with singly biotinylated antigen (which issubsequently termed a streptavidin microaggregate as described below).Streptavidin pre-loading can increase the effective antigenconcentration up to 500-fold, and is useful for isolating weak antigenbinders that bind specifically to the antigen. Employment ofstreptavidin microaggregates is useful for isolating antibodies rangingin affinity from very weak to moderate (Kd greater than about 200 nM)affinities. Furthermore, biotinylated epitopes can be pre-reacted withstreptavidin-fluorophore at room temperature for 10 to 15 minutes inorder to create microaggregates prior to contacting cell populations.The microaggregates are subsequently allowed to contact cellssimultaneously for 15 to 30 minutes prior to addition of secondaryreagents, such as anti-human IgG-fluorophore conjugates. In oneexperimental approach, cells are centrifuged at 1500×g for 5 minutes andresuspended in a small volume (typically 500 μL to 1 mL) of DAPI (PBS,1% BSA, 2 μg/mL DAPI). In a second approach termed “homogeneous assayconditions,” cells are resuspended directly in DAPI into whichantigen-streptavidin microaggregate and goat-anti-human IgG-fluorophoreare added. This second approach is particularly desirable for moreweakly interacting antibodies (Kd greater than about 200 nM), whereminimizing dissociation time may be more relevant.

At higher affinities (with Kd>10 nM, but less than about 100 nM),libraries are more easily screened directly for improved affinity byincubating the library with monomeric binding peptide or full lengthtarget protein under equilibrium binding conditions at a concentrationof binding peptide that is ideally less than the Kd of the starting(wild type) interaction (apparent Kds can be readily determined by aseries of analytical FACS experiments conducted with a range of antigenconcentrations, ahead of a sort). Under these conditions, cells thatpossess antibodies and binding proteins with higher affinities willpossess significantly more fluorescently labeled binding peptide thanweaker binders, allowing the most fluorescent cells in the population tobe easily selected for further optimization. Typically, FACS sortinggates can be established that select about the top 0.5% to about 0.1% ofcells. In one non-limiting method, about the top 0.2% of cells areselected.

As recognized by Boder and Wittrup (Biotechnol. Prog. (1998) 14 55-62),the screening of very high affinity protein-ligand interactions (Kd<10mM) can be accomplished by screening for decreased off-rate rather thandirectly for affinity. In this approach, cells are labeled to saturationwith fluorescent binding peptide, followed by addition of an excess ofnon-fluorescent ligand. Cell associated fluorescence decaysexponentially with time approaching a background level and thedissociation reaction is stopped after a fixed duration, usually byextensive dilution with cold buffer. The duration of the competitionreaction determines the difference in observed fluorescence fordifferent library clones and, thus, determines the range of kineticimprovements likely to be selected from the library. For a competitivedissociation reaction, the presence of excess non-fluorescent ligand canyield an effective forward reaction rate of zero. Mean fluorescenceintensity at a given time after the initiation of the competitionreaction is a function of the off-rate (K_(off)). (VanAntwerp & Wittrup(2000) Biotechnol. Prog. 16 31-37; Boder et al. (2000) PNAS 9710701-10705; and Foote and Eisen (2000) PNAS 97 10679-10681). Cells inthe population that express antibodies with improved affinities and morestable binding can be systematically identified by progressivelyincreasing the length of time for the competition reaction, and thenselecting the most fluorescent cells remaining in the population forfurther optimization.

Under these conditions, cells that possess surface displayed antibodiesand binding proteins with higher affinities will exhibit significantlymore bead or streptavidin-biotinylated antigen microaggregate bindingcompared to cells that express proteins with little or no binding. Themost fluorescently labeled cells (displaying proteins with the highestaffinity) can then be separated from the rest of the cells in thepopulation using standard FACS sorting protocols, as described, forexample, in Example 9.

Once a selected cell population has been created that expresses aprotein that exhibits reproducible binding to a binding peptide, it canbe characterized with two or more intact proteins to confirm that theantibodies or binding proteins exhibit the desired pattern ofcross-reactivity and/or specificity (e.g., to both mouse and humanvariants of the protein of interest), or to two different members of arelated gene family, but not to an unrelated, or more distantly related,protein.

In one embodiment, this can be accomplished using multi-parameter FACSusing two or more proteins species labeled with two differently coloreddetectable. tags (e.g., FITC and phycoerythrin) which can besimultaneously analyzed in a flow cytometer. Using this approach, it ispossible to identify cells that display binding to only one protein, orare capable of binding to both proteins. The population of cells thatexhibits the required dual specific binding can be selected by the FACSoperator based upon the number of cells sorted and the percentage ofcells identified that exhibit polyspecificity. As described previously,these selected cells can subsequently be allowed to recover and grownusing standard culture conditions for a number of days until thepopulation has reached a reasonable number to enable either a subsequentround of FACS sorting, analysis, cell banking, or to determine sequenceinformation.

Selected binders from the library can be further characterized asdescribed herein, and the sequence of the antibody or binding proteindetermined after PCR of cellular DNA, RT-PCR of RNA isolated from theselected cell population, or episome rescue.

Candidate antibodies and binding proteins can be iteratively subjectedto rounds of hypermutation and selection in order to evolve populationsof cells expressing antibodies or binding proteins with enhanced bindingproperties as described herein. Cells that preferentially and/orselectively bind to the binding peptide with a higher affinity areselected and allowed to expand. If needed, another round of mutagenesisis repeated and, again, cells that exhibit improved, selective, and highaffinity binding, are retained for further propagation and growth. Thenew improved variants obtained can be further characterized as describedherein, and the sequence of the heavy and light chains determined afterRT-PCR or episome rescue.

Mutations that are identified in the first one, two or three rounds ofhypermutation/selection can be recombined combinatorially into a set ofnew templates within the original parental backbone context, and all, ora subset of the resulting templates, can be subsequently transfectedinto cells which are then selected by FACS sorting. The bestcombination(s) of mutations are thus isolated and identified, and eitherused in a subsequent round of hypermutation/selection, or if the newlyidentified template(s) demonstrate sufficiently potent affinity, areused instead in experiments for further functional characterization.

In another embodiment, FACS can be used to screen a library of cellsexpressing intracellular proteins that are undergoing, or haveundergone, SHM mediated diversity creation. In this approach, a membranepermeable fluorogenic, or florescent reagent is used and firstpre-incubated with the library of cells to allow uptake and conversionof the reagent. The FACS instrument is able to separate the highactivity protein members of the library, which are able to convert agreater percentage of the reagent and are more fluorescent than cellscomprising lower activity members. (See, e.g., Farinas, Comb. Chem. HighThroughput Screen. (2006) 9: (4) 321-328).

Fluorescent moieties to be detected include, but are not limited to,compounds such as fluorescein (commonly called FITC), phycobiliproteinssuch as phycoerythrin (PE) and allophycocyanin (APC) (Kronick, M. N. J.Imm. Meth. 92:1-13 (1986)), fluorescent semiconductor nanocrystals suchas Quantum dot (QDot) bioconjugates for ultrasensitive nonisotopicdetection (Chan W C, Nie S. Science 281: 2016-8 (1998)), and coumarinderivatives such as Fluorescent Acylating Agents derived from7-Hydroxycoumarin.

Fluorescence can also reported from fluorescent proteins such as TealFluorescent Protein (TFP), from chemical stains of cellular componentssuch as DAPI bound to DNA, from fluorescent moieties covalentlyconjugated to antibodies that recognize cellular products, fromfluorescent moieties covalently conjugated to ligands of cellularreceptors, and from fluorescent moieties covalently conjugated tosubstrates of cellular enzymes.

Cells stained with membrane impermeant reporters, such as antibodies,can be sorted for subsequent processing to recover components such asgenes, episomes, or proteins of interest. Cells stained for surfaceexpression components or stained with cell membrane permeant reporterscan also be sorted intact for propagation.

2. Affinity Separation

Affinity separation based on the use microparticles enables theseparation of surface displayed proteins based on affinity to a specificcompound or sequence of interest. This approach is rapid, can easily bescaled up, and can be used iteratively with living cells.

Paramagnetic polystyrene microparticles are commercially available(Spherotech, Inc., Libertyville, Ill.; Invitrogen, Carlsbad, Calif.)that couple compounds or peptides to microparticle surfaces that havebeen modified with functional groups or coated with various antibodiesor ligands such as, for example, avidin, streptavidin or biotin.

In one aspect paramagnetic beads can be used in which the paramagneticproperty of microparticles allows them to be separated from solutionusing a magnet. The microparticles can be easily re-suspended whenremoved from the magnet thereby enabling the selective separation ofcells that find to the attached probe.

In one embodiment, peptides can be coupled to paramagnetic polystyrenemicroparticles coated with a polyurethane layer in a tube. The hydroxylgroups on the microparticle surface are activated by reaction withp-toluensulphonyl chloride (Nilsson κ and Mosbach K. “p-Toluenesulfonylchloride as an activating agent of agarose for the preparation ofimmobilized affinity ligands and proteins.” Eur. J. Biochem. 1980:112:397-402). The resulting sulphonyl ester can subsequently reactcovalently with peptide amino or sulfhydryl groups. The peptides arequickly absorbed onto the surface of the activated microparticlesfollowed by the formation of covalent amine bonds with furtherincubation. The microparticles (2⁰⁹ microparticles/milliliter) arewashed two times by placing the tube containing 1 milliliter (ml) ofmicroparticles on a magnet, allowing the microparticles to migrate tothe magnet side of the tube, removing the supernatant, and re-suspendingthe microparticles in 1 ml of 100 millimolar (mM) borate buffer, pH 9.5.After washing, the microparticles are re-suspended in 100 mM boratebuffer, pH 9.5 at a concentration of 109 microparticles/ml. Elevennanomoles of peptide are added to the microparticles and themicroparticle/peptide mixture is vortexed for 1 minute to mix. Themicroparticles are incubated with peptides at room temperature for atleast 48 hours with slow tilt rotation. To ensure an optimal orientationof the peptide on the microparticles, bovine serum albumin (BSA) isadded to the microparticle/peptide mixture to a final concentration of0.1% (weight/volume) after incubation has proceeded for 10 minutes.After incubation, the tube containing the microparticle/peptide mixtureis placed on the magnet until the microparticles migrate to the magnetside of the tube. The supernatant is removed and the microparticles arewashed four times with 1 ml phosphate buffered saline solution (PBS), pH7.2 containing 1% (weight/volume) BSA. Finally, the microparticles arere-suspended in 1 ml PBS solution, pH 7.2 containing 1% (weight/volume)BSA.

Alternatively, paramagnetic polystyrene microparticles containingsurface carboxylic acid can be activated with a carbodiimide followed bycoupling to a peptide, resulting in a stable amide bond between aprimary amino group of the peptide and the carboxylic acid groups on thesurface of the microparticles (Nakajima N and Ikade Y, Mechanism ofamide formation by carbodiimide for bioconjugation in aqueous media,Bioconjugate Chem. 1995, 6(1), 123-130; Gilles M A, Hudson A Q andBorders C L Jr, Stability of water-soluble carbodiimides in aqueoussolution, Anal Biochem. 1990 Feb. 1; 184(2):244-248; Sehgal D and VijayI K, a method for the high efficiency of water-solublecarbodiimide-mediated amidation, Anal Biochem. 1994 April; 218(1):87-91;Szajani B et al, Effects of carbodiimide structure on the immobilizationof enzymes, Appl Biochem Biotechnol. 1991 August; 30(2):225-231). Themicroparticles (2⁹ microparticles/milliliter) are washed twice with 1 mlof 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 for 10 minutes withslow tilt rotation at room temperature. The washed microparticles arere-suspended in 700 microliters (μL) 25 mM 2-[N-morpholino]ethanesulfonic acid, pH 5 followed by the addition of 21 nanomoles of peptidere-suspended in 25 mM 2-[N-morpholino]ethane sulfonic acid, pH 5 to themicroparticle solution. The microparticle/peptide mixture is mixed byvortexing and incubated with slow tilt rotation for 30 minutes at roomtemperature. After this first incubation, 300 μL of ice-cold 100milligram (mg)/mL 1-ethyl-3-(3-dimethylaminopropyl) carbodiimidehydrochloride re-suspended in 25 mM 2-[N-morpholino]ethane sulfonicacid, pH 5 is added to the peptide/microparticle mixture and incubatedovernight at 4° Celsius with slow tilt rotation. The peptide-coupledmicroparticles are washed four times with 1 ml 50 mM Tris pH 7.4/0.1%BSA for 15 minutes at room temperature with slow tilt rotation. Afterwashing, the peptide-coupled microparticles are re-suspended at aconcentration of 1⁹ microparticles/ml in PBS solution, pH 7.2 containing1% (weight/volume) BSA.

Another option is to couple biotinylated peptides to paramagneticpolystyrene microparticles whose surfaces have been covalently linkedwith a monolayer of streptavidin. Briefly, one ml of the streptavidinmicroparticles are transferred to a microcentrifuge tube and washed fourtimes by placing the tube on a magnet and allowing the microparticles tocollect on the magnet side of the tube. The solution is then removed andthe microparticles are gently re-suspended in 1 ml of PBS solution, pH7.2 containing 1% (weight/volume) BSA. After the final wash, themicroparticles are re-suspended in 1 ml of PBS solution, pH 7.2containing 1% (weight/volume) BSA; and 33 picomoles of biotinylatedpeptide are added to the microparticle solution. Themicroparticle/peptide solution is incubated for 30 minutes at roomtemperature with slow tilt rotation. After coupling, the unboundbiotinylated peptide is removed from the microparticles by washing fourtimes with PBS solution, pH 7.2 containing 1% (weight/volume) BSA. Afterthe final wash, the microparticle/peptide mixture is re-suspended to afinal bead concentration of 1⁹ microparticles/ml. (Argarana C E, Kuntz ID, Birken S, Axel R, Cantor C R. Molecular cloning and nucleotidesequence of the streptavidin gene. Nucleic Acids Res. 1986;14(4):1871-82; Pahler A, Hendrickson W A, Gawinowicz Kolks M A, AraganaC E, Cantor C R. Characterization and crystallization of corestreptavidin. Biol Chem 1987:262(29):13933-7)

The identification, selection and use of specific peptide sequences foruse in the present inventions is disclosed in commonly owned priorityapplication No. 60/995,970 (Attorney docket no. 33547-708.101), filedSep. 28, 2007.

XI. Pharmaceutical Formulations

Pharmaceutical formulations comprising a protein of interest, e.g., anantibody, identified by the methods of the present invention can beprepared for storage by mixing the protein having the desired degree ofpurity with optional physiologically acceptable carriers, excipients orstabilizers (Remington's Pharmaceutical Sciences 16th edition, Osol, A.Ed. (1980)), in the form of lyophilized formulations or aqueoussolutions. Acceptable carriers, excipients, or stabilizers are nontoxicto recipients at the dosages and concentrations employed, and includebuffers such as phosphate, citrate, and other organic acids;antioxidants including ascorbic acid and methionine; preservatives (suchas octadecyldimethylbenzyl ammonium chloride; hexamethonium chloride;benzalkonium chloride, benzethonium chloride; phenol, butyl or benzylalcohol; alkyl parabens such as methyl or propyl paraben; catechol;resorcinol; cyclohexanol; 3-pentanol; and m-cresol); low molecularweight (less than about 10 residues) polypeptides; proteins, such asserum albumin, gelatin, or immunoglobulins; hydrophilic polymers such aspolyvinylpyrrolidone; amino acids such as glycine, glutamine,asparagine, histidine, arginine, or lysine; monosaccharides,disaccharides, and other carbohydrates including glucose, mannose, ordextrins; chelating agents such as EDTA; sugars such as sucrose,mannitol, trehalose or sorbitol; salt-forming counter-ions such assodium; metal complexes (e.g., Zn-protein complexes); and/or non-ionicsurfactants such as TWEEN®, PLURONICS® or polyethylene glycol (PEG).

The formulation described herein can also contain more than one activecompound as necessary for the particular indication being treated. Suchmolecules are suitably present in combination in amounts that areeffective for the purpose intended.

In one embodiment, the pharmaceutical formulations can comprise anantibody identified by the methods described herein. In certainembodiments, the pharmaceutical formulation can be in a microcapsuleprepared, for example, by coacervation techniques or by interfacialpolymerization, for example, hydroxymethylcellulose orgelatin-microcapsule and poly-(methylmethacylate) microcapsule,respectively, in colloidal drug delivery systems (for example,liposomes, albumin microspheres, microemulsions, nano-particles andnanocapsules) or in macroemulsions. Such techniques are disclosed inRemington's Pharmaceutical Sciences 16th edition, Osol, A. Ed. (1980).

In still other embodiments, sustained-release preparations can beprepared. Suitable examples of sustained-release preparations includesemi-permeable matrices of solid hydrophobic polymers containing theprotein, which matrices are in the form of shaped articles, e.g., films,or microcapsule. Examples of sustained-release matrices includepolyesters, hydrogels (for example, poly(2-hydroxyethyl-methacrylate),or poly(vinylalcohol)), polylactides (U.S. Pat. No. 3,773,919),copolymers of L-glutamic acid and gamma. ethyl-L-glutamate,non-degradable ethylene-vinyl acetate, degradable lactic acid-glycolicacid copolymers such as the Lupron Depot® (injectable microspherescomposed of lactic acid-glycolic acid copolymer and leuprolide acetate),and poly-D-(−)-3-hydroxybutyric acid. While polymers such asethylene-vinyl acetate and lactic acid-glycolic acid enable release ofmolecules for over 100 days, certain hydrogels release proteins forshorter time periods. When encapsulated proteins remain in the body fora long time, they can denature or aggregate as a result of exposure tomoisture at 37° C., resulting in a loss of biological activity andpossible changes in immunogenicity. Rational strategies can be devisedfor stabilization depending on the mechanism involved. For example, ifthe aggregation mechanism is discovered to be intermolecular S—S bondformation through thio-disulfide interchange, stabilization can beachieved by modifying sulfhydryl residues, lyophilizing from acidicsolutions, controlling moisture content, using appropriate additives,and developing specific polymer matrix compositions.

XII. Non-Therapeutic Uses

The proteins of interest, e.g., antibodies, identified by the methods ofthe present invention can be used non-therapeutic agents, for example,as affinity purification agents. In such an embodiment, a protein ofinterest is immobilized on a solid phase such a Sephadex resin or filterpaper, using methods well known in the art. The immobilized protein iscontacted with a sample containing the target of interest (or fragmentthereof) to be purified, and thereafter the support is washed with asuitable solvent that will remove substantially all the material in thesample except the target protein, which is bound to the immobilizedantibody. Finally, the support is washed with another suitable solvent,such as glycine buffer, pH 5.0, which will release the target protein.

Proteins identified by the methods of the present invention can also beuseful in diagnostic assays for the targeted protein, e.g., detectingits expression in specific cells, tissues, or serum. Such diagnosticmethods can be useful in cancer diagnosis.

For diagnostic applications, the proteins will typically be labeled witha detectable moiety. In certain embodiments, the detectable moiety canbe selected from the following categories: (a) Radioisotopes, such as³⁵S, ¹⁴C, ¹²⁵I, ³H, and ¹³¹I. The antibody can be labeled with theradioisotope using the techniques described in Current Protocols inImmunology, Volumes 1 and 2, Coligen et al., Ed. Wiley-Interscience, NewYork, N.Y., Pubs. (1991) for example and radioactivity can be measuredusing scintillation counting; (b) Fluorescent labels such as rare earthchelates (europium chelates) or fluorescein and its derivatives,rhodamine and its derivatives, dansyl, Lissamine, phycoerythrin andTexas Red are available; (c) enzyme-substrate labels.

Various enzyme substrate labels are known in the art and U.S. Pat. No.4,275,149 provides a review of some of these. The enzyme generallycatalyzes a chemical alteration of the chromogenic substrate which canbe measured using various techniques. For example, the enzyme cancatalyze a color change in a substrate, which can be measuredspectrophotometrically. Alternatively, the enzyme can alter thefluorescence or chemiluminescence of the substrate. Techniques forquantifying a change in fluorescence are described above. Thechemiluminescent substrate becomes electronically excited by a chemicalreaction and can then emit light which can be measured (using achemiluminometer, for example) or donates energy to a fluorescentacceptor. Examples of enzymatic labels include luciferases (e.g.,firefly luciferase and bacterial luciferase; U.S. Pat. No. 4,737,456),luciferin, 2,3-dihydrophthalazinediones, malate dehydrogenase, urease,peroxidase such as horseradish peroxidase (HRPO), alkaline phosphatase,β-galactosidase, glucoamylase, lysozyme, saccharide oxidases (e.g.,glucose oxidase, galactose oxidase, and glucose-6-phosphatedehydrogenase), heterocyclic oxidases (such as uricase and xanthineoxidase), lactoperoxidase, microperoxidase, and the like. Techniques forconjugating enzymes to antibodies are described in O'Sullivan et al.,Methods for the Preparation of Enzyme-Antibody Conjugates for use inEnzyme Immunoassay, in Methods in Enzymol. (ed J. Langone & H. VanVunakis), Academic press, New York, 73:147 166 (1981).

In certain embodiments, enzyme-substrate combinations can include, forexample: (i) Horseradish peroxidase (HRPO) with hydrogen peroxidase as asubstrate, wherein the hydrogen peroxidase oxidizes a dye precursor(e.g., orthophenylene diamine (OPD)); (ii) alkaline phosphatase (AP)with para-Nitrophenyl phosphate as chromogenic substrate; and (iii)β-D-galactosidase (β-D-Gal) with a chromogenic substrate (e.g.,p-nitrophenyl-β-D-galactosidase) or fluorogenic substrate4-methylumbelliferyl-β-D-galactosidase.

Numerous other enzyme-substrate combinations are available to thoseskilled in the art. For a general review of these, see U.S. Pat. Nos.4,275,149 and 4,318,980.

The proteins identified by the methods of the present invention can beemployed in any known assay method, such as competitive binding assays,direct and indirect sandwich assays, and immunoprecipitation assays.Zola, Monoclonal Antibodies: A Manual of Techniques, pp. 147 158 (CRCPress, Inc. 1987).

The antibodies can also be used for in vivo diagnostic assays.Generally, the antibody is labeled with a radio nuclide (such as ¹¹¹In,¹⁴C, ¹³¹I, ¹²⁵I, ³H, ³²P or ³⁵S) so that the tumor can be localizedusing immunoscintiography.

XIII. Therapeutic Uses

For therapeutic applications, the proteins, including but not limited toantibodies, identified by the methods of the present invention can beadministered to a mammal, preferably a human, in a pharmaceuticallyacceptable dosage form such as those discussed above, including thosethat can be administered to a human intravenously as a bolus or bycontinuous infusion over a period of time, by intramuscular,intraperitoneal, intra-cerebrospinal, subcutaneous, intra-articular,intrasynovial, intrathecal, oral, topical, or inhalation routes.Proteins including but not limited to antibodies identified by themethods of the present invention also can be suitably administered byintra tumoral, peritumoral, intralesional, or perilesional routes, toexert local as well as systemic therapeutic effects. The intraperitonealroute is expected to be particularly useful, for example, in thetreatment of ovarian tumors.

For the prevention or treatment of disease, the appropriate dosage of atherapeutic protein will depend on the type of disease to be treated, asdefined above, the severity and course of the disease, whether theprotein is administered for preventive or therapeutic purposes, previoustherapy, the patient's clinical history and response to the protein, andthe discretion of the attending physician. The antibody is suitablyadministered to the patient at one time or over a series of treatments.

XIV. Databases

The invention includes methods of producing computer-readable databasescomprising the sequence and identified mutations of certain proteins,including, but not limited to, sequences of binding domains, or activesites, as well as their binding characteristics, activity, stabilitycharacteristics and three-dimensional molecular structure. Specificallyincluded in the present invention is the use of such a database to aidin the design and optimization of a protein of interest, based on adatabase of mutations created from the protein of interest, or relatedproteins or portions thereof.

In other embodiments, the databases of the present invention cancomprise mutations of a protein or proteins that have been identified byscreening to bind to a specific target, or other representations of suchproteins such as, for example, a graphic representation or a name.

By “database” is meant a collection of retrievable data. The inventionencompasses machine readable media embedded with or containinginformation regarding the amino acid and nucleic structure of a proteinor proteins, such as, for example, its sequence, structure, and theactivity or binding activity, as described herein. Such information canpertain to subunits, domains, and/or portions thereof such as, forexample, portions comprising active sites, accessory binding sites,and/or binding pockets in either liganded (bound) or unliganded(unbound) forms.

Alternatively, the information can be that of identifiers whichrepresent specific structures found in a protein. As used herein,“machine readable medium” refers to any medium that can be read andaccessed directly by a computer or scanner. Such media can take manyforms, including but not limited to, non-volatile, volatile andtransmission media. Non-volatile media, i.e., media that can retaininformation in the absence of power, includes a ROM. Volatile media,i.e., media that cannot retain information in the absence of power,includes a main memory.

Transmission media includes coaxial cables, copper wire and fiberoptics, including the wires that comprise the bus. Transmission mediacan also take the form of carrier waves; i.e., electromagnetic wavesthat can be modulated, as in frequency, amplitude or phase, to transmitinformation signals. Additionally, transmission media can take the formof acoustic or light waves, such as those generated during radio waveand infrared data communications.

Such media also include, but are not limited to: magnetic storage media,such as floppy discs, flexible discs, hard disc storage medium andmagnetic tape; optical storage media such as optical discs or CD-ROM;electrical storage media such as RAM or ROM, PROM (i.e., programmableread only memory), EPROM (i.e., erasable programmable read only memory),including FLASH-EPROM, any other memory chip or cartridge, carrierwaves, or any other medium from which a processor can retrieveinformation, and hybrids of these categories such as magnetic/opticalstorage media. Such media further include paper on which is recorded arepresentation of the amino acid or polynucleotide sequence, that can beread by a scanning device and converted into a format readily accessedby a computer or by any of the software programs described herein by,for example, optical character recognition (OCR) software. Such mediaalso include physical media with patterns of holes, such as, forexample, punch cards and paper tape.

Specifically included in the present invention is the transmission ofdata from the data base via transmission media to third party site toaid in the design and optimization of a protein of interest.

A variety of data storage structures are available for creating acomputer readable medium having recorded thereon the amino acid orpolynucleotide sequences of the invention or portions thereof and/oractivity data. The choice of the data storage structure can be based onthe means chosen to access the stored information. All formatrepresentations of the amino acid or polynucleotide sequences describedherein, or portions thereof, are contemplated by the present invention.By providing computer readable medium having stored thereon thesequences of the invention, one can routinely access the SHM mediatedchanges in amino acid or polynucleotide sequence and related informationfor use in modeling and design programs, to create improved proteins.

A computer can be used to display the sequence of the protein or peptidestructures, or portions thereof, such as, for example, portionscomprising active sites, accessory binding sites, and/or bindingpockets, in either liganded or unliganded form, of the presentinvention. The term “computer” includes, but is not limited to,mainframe computers, personal computers, portable laptop computers, andpersonal data assistants (“PDAs”) which can store data and independentlyrun one or more applications, i.e., programs. The computer can include,for example, a machine readable storage medium of the present invention,a working memory for storing instructions for processing themachine-readable data encoded in the machine readable storage medium, acentral processing unit operably coupled to the working memory and tothe machine readable storage medium for processing the machine readableinformation, and a display operably coupled to the central processingunit for displaying the structure coordinates or the three-dimensionalrepresentation.

The computers of the present invention can also include, for example, acentral processing unit, a working memory which can be, for example,random-access memory (RAM) or “core memory,” mass storage memory (forexample, one or more disk drives or CD-ROM drives), one or morecathode-ray tube (“CRT”) display terminals or one or more LCD displays,one or more keyboards, one or more input lines, and one or more outputlines, all of which are interconnected by a conventional bi-directionalsystem bus. Machine-readable data of the present invention can beinputted and/or outputted through a modem or modems connected by atelephone line or a dedicated data line (either of which can include,for example, wireless modes of communication). The input hardware canalso (or instead) comprise CD-ROM drives or disk drives. Other examplesof input devices are a keyboard, a mouse, a trackball, a finger pad, orcursor direction keys. Output hardware can also be implemented byconventional devices. For example, output hardware can include a CRT, orany other display terminal, a printer, or a disk drive. The CPUcoordinates the use of the various input and output devices, coordinatesdata accesses from mass storage and accesses to and from working memory,and determines the order of data processing steps. The computer can usevarious software programs to process the data of the present invention.Examples of many of these types of software are discussed throughout thepresent application.

EXAMPLES

While a number of embodiments of the present invention have been shownand described herein, such embodiments are provided by way of exampleonly. It should be understood that various alternatives to theembodiments of the invention described herein may be employed inpracticing the invention. It is intended that the following claimsdefine the scope of the invention and that methods and structures withinthe scope of these claims and their equivalents be covered thereby.

Example 1 Creation of Synthetic Polynucleotides Encoding Blasticidin

By decreasing the likelihood of somatic hypermutation in a vectorelement, such as a selectable marker, an enzyme involved in SHM, or areporter gene, the vector and system for exerting and tracking SHMbecomes more stable, thereby enabling somatic hypermutation to be moreeffectively targeted to a polynucleotide or library of polynucleotidesof interest.

A. Polynucleotide Design

In general, sequences are engineered for SHM using the teachingdescribed herein, and as elaborated in sections III and IV of U.S.application No. 60/902,414, entitled “Systems for SomaticHypermutation.” In the following examples, sequence optimization isbased on the hot spot and cold spot definitions listed herein in Table6), and using the computer program SHMredesign:

Using this program, every position within the sequence is annotated witheither a ‘+’, ‘−’, or ‘.’ symbol to designate whether it is desired toobtain a hotter, a colder, or a neutral change in SHM susceptibility atthat specific position, where ‘+’ designates a hot spot, ‘−’ cold spot,and ‘.’ a neutral position. For example, the following input sequencefor blasticidin is used to identify SHM resistant versions at everyposition of the blasticidin gene.

By comparison, the following input file is used to identify hotterversions of the blasticidin gene that are more susceptible to SHM atevery position of the gene.

As described previously, during this process, all nucleotide sequencesover a 9 base region consistent with the wild type protein's amino acidsequence are enumerated and scored for the number of hot spots, coldspots, CpG motifs, codon usage, and stretches of the same nucleotide.The program then determines whether it is possible to replace any randomsequence with a hotter, a colder, or a neutral polynucleotide tileencoding the same three amino acids.

As shown in FIG. 7, this approach, as applied to canine AID quickly,(within a few hundred tile substitutions), converges to identify a coldoptimized canine AID new sequence, which differs from the originalsequence through the substitution 15-20% of the nucleotide sequence. Themajority of changes occur early in the iterative cycle and are usuallycomplete after about 500 iterations. As one might expect, larger genesrequire a larger number of iterations to reach a fully optimizedsequence. Routinely, the use of 2000 to 3000 iterations is more thansufficient for the majority of genes.

Analysis of a number of unmodified genes at random demonstrates thatmost mammalian genes use codons that create on average about 9 to 15cold spots per 100 nucleotides, and with a median density of about 13.8cold spots/100 nucleotides, and have a hot spot density of between about7 to 13 hot spots per 100 nucleotides, with a median density of about8.3 hot spots per 100 nucleotides.

The initial starting sequence, as well as the frequency of hot spots,cold spots and CpGs for the unmodified, blasticidin gene are shown inFIG. 8.

1. Cold Blasticidin

An optimized sequence for a SHM resistant (cold) version of blasticidincreated using this approach is shown in FIG. 9, together with theresulting changes in frequency of hot spots and cold spots. Optimizationof the blasticidin sequence to make the sequence more resistant tosomatic hypermutation resulted in an increase of 188% in number of coldspots (an increase of 73), and reduced the number of hot spots by 57% (adecrease of 15). Overall, the frequency of cold spots increased to anaverage density of about 28 cold spots per 100 nucleotides from aninitial density of about 15 cold spots per 100 nucleotides, and theoverall frequency of hot spots decreased from about 9 hot spots per 100nucleotides, in the unmodified gene to about 5 hot spots per 100nucleotides in the SHM resistant form.

2. Hot Blasticidin

An optimized sequence for a SHM susceptible version of blasticidincreated using this approach is shown in FIG. 10, together with theresulting changes in frequency of hot spots and cold spots. Optimizationof the blasticidin sequence to make the sequence more susceptible tosomatic hypermutation resulted in an increase of about 197% in number ofhot spots (an increase of 34), and reduced the number of cold spots byabout 56% (a decrease of 26). Overall, the frequency of hot spotsincreased to an average density of about 17 hot spots per 100nucleotides from an initial density of about 9 hot spots per 100nucleotides, and the overall frequency of cold spots decreased fromabout 15 cold spots per 100 nucleotides in the unmodified gene to about9 cold spots per 100 nucleotides in the SHM susceptible form.

B. Cloning and Analysis

After final review to ensure that the synthetic polynucleotide sequenceis free of extraneous restriction sites, the complete polynucleotidesequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into oneof DNA2.0's cloning vectors (see Table 10 below), and sequenced toconfirm correct synthesis.

TABLE 10 DNA2.0 source restriction sites vector that insert Constructplasmid (5′, 3′) was cloned into cold TFP pJ15 Sac1, BsrG1 AB136 hot TFPpJ15 Sac1, BsrG1 AB102 GFP* stop (Y82stop) pJ31 Sac1, BsrG1 AB105 coldhygromycin pJ2 NgoMIV, Xba1 AB179, AB163 native puromycin pJ51 NgoMIV,Xba1 AB150, AB161 cold blasticidin pJ13 NgoMIV, Xba1 AB102, AB153 coldAID pJ45 Sac1, BsrG1 AB135, AB174 Heavy chains scaffolds H1 pJ31 H2-H9pJ51 SgrAI, EagI ANA320 Kappa Light chains K1 pJ31 K2-K4 pJ51 SbfI,BsmBI XX13 Lambda Light chains L1 pJ31 L2-L5 pJ51 SbfI, BbsI XX13

Other elements, for example E-box motifs or Ig enhancer elements, arecreated by either oligo synthesis or PCR amplification as described inU.S. Patent application No. 60/902,414, entitled “Somatic HypermutationSystems,” filed on Feb. 20, 2007, and specifically incorporated hereinin its entirety.

To test the functionality of the new synthetic inserts, coding regionsare excised from DNA2.0 source vectors using restriction enzymes aslisted in Table 10 above, and inserted into expression vectors (Table10) using standard recombinant molecular biological techniques.Insertion of selection markers (i.e., cold blasticidin, cold hygromycin,and native puromycin) into the AB series of vectors places them downstream of the EMCV IRES sequence (AB150, AB102, AB179; see FIG. 17A) ordownstream of the pSV promoter (AB161, AB153, AB163; see FIG. 17B).

To test functional activity of the optimized synthetic genes, Hek 293cells are plated at 4×10⁵/well, in 6-well microtiter dish. After 24hours, transfections are performed using Fugene6 reagent from RocheApplied Sciences (Indianapolis, Ind.) at a reagent-to-DNA ratio of 3μL:1 μg DNA per well. This ratio is also maintained for transfectionswith multiple plasmids. Transfections are carried out in accordance withmanufacturer's protocol.

To determine the relative stability/susceptibility of each construct tosomatic hypermutation, stable cell lines of each transfected cellpopulation are created, and tested to determine the relative speed bywhich they accumulate SHM mediated mutations. Because the majority ofthese mutations result in a loss of function, relative mutagenesis loadare conveniently measured as a loss of fluorescence via FACS (see belowand Example 2).

FACS Analysis. Prior to FACS analysis, cells are harvested bytrypsinization, washed twice in PBS containing 1% w/v BSA, andre-suspended in 200 μl PBS/1% BSA containing 2 ng/ml DAPI. Cells areanalyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nmlaser excitation. Up to one million cells per sample are acquired. DAPIfluorescence is measured through a 460/50 bandpass filter. GFPfluorescence is measured through a 528/38 bandpass filter. Percent GFPexpression is reported as percentage of DAPI excluding live cells withno detectable GFP fluorescence above cellular background.

Reversion assays to test for function of the canine AID gene. GFP* (GFPwith a stop codon introduced by site directed mutagenesis at position 82[Y82stop]) is co-transfected with AB174 (cold canine AID), and cells areanalyzed by flow cytometry 3 days post transfection, placed underantibiotic selection and analyzed further by flow cytometry every otherday for 13-15 days.

Antibiotic selections. Antibiotic concentrations used in the selectionof Hek 293 cells are determined empirically by performing a kill curve(i.e., determining the minimal concentration of antibiotic that killsall un-transfected—and thus antibiotic sensitive-cells). At 3 days posttransfection, cells are plated at 4×10⁵/well and selected at thefollowing concentrations: 1.5 μg/mil puromycin (Clontech, Mountain View,Calif.); 16 μg/mL blasticidin (Invitrogen, Carlsbad, Calif.); and 360μg/mL hygromycin (Invitrogen, Carlsbad, Calif.).

Resistance marker genes are tested to determine functionality bytransfection of the appropriate expression plasmid (i.e. AB102 forblasticidin, AB179 for hygromycin) in Hek 293 cells based on theirability to promote drug resistance cell growth in the presence of 16μg/mL blasticidin (Invitrogen, Carlsbad, Calif.); and 360 μg/mLhygromycin (Invitrogen, Carlsbad, Calif.) for two weeks.

Transfection of the AB102 containing cold blasticidin resulted in thecreation of drug resistant colonies of transfected hek 293 cells atcomparable rates as the wild type gene.

Example 2 Creation of Synthetic Polynucleotides Encoding EnzymesInvolved in SHM Cytidine Deaminase (AID)

Analysis of sequence variations in cytidine deaminase (AID) betweenmammalian species (rat, chimpanzee, mouse, human, dog, cow, rabbit,chicken, frog, zebra fish, fugu and tetraodon (puffer fish)) as comparedto humans demonstrates that organisms as distantly related as human andfrog display a surprisingly high (70%) sequence identity, and >80%sequence similarity. In addition, it has been shown that AID from otherorganisms can be substituted for human AID in somatic hypermutation(SHM), and that all mammalian species of AID are functionallyequivalent.

Shown in FIG. 11 is a comparison of human AID with other terrestrialAIDs in order to identify a potential beginning construct for SHM invivo. The figure provides a sequence alignment of AID from human(H_sap/1-198), mouse (M_musc/1-198), canine (C_fam/1-198), rat(R_norv/1-199), and chimpanzee (P_trog/1-199). FIG. 15 illustrates thesequence identity between human, canine and mouse AID proteins

As shown by FIG. 11, canine AID has overall 94% amino acid identity tohuman and mouse AID and, thus, is selected as the starting point forcodon optimization. To optimize codon usage, the canine amino acidsequences are reverse translated and then iteratively optimized.

AID is known to contain a nuclear export signal, which is containedwithin the C-terminal 10 amino acids (McBride et al., Somatichypermutation is limited by CRM1-dependent nuclear export ofactivation-induced deaminase, J Exp Med. 2004 May 3; 199(9):1235-44; Itoet al., Activation-induced cytidine deaminase shuttles between nucleusand cytoplasm like apolipoprotein B mRNA editing catalytic polypeptide1, PNAS 2004 Feb. 17; 101(7):1975-80.) For purposes of the experimentsdescribed below, the canine AID contains a leucine to alanine mutationat position 198, while the human AID construct retains the unmutated,intact nuclear export signal.

A. Polynucleotide Design

As described in Example 1, SHM sequence optimization is completed usingthe computer program SHMredesign, based on the hot spot and cold spotdefinitions listed in Table 6; the resulting hot and cold versions ofcanine AID are shown in FIG. 13 and FIG. 14 respectively. The startingsequence for canine AID is shown in FIG. 12, together with the initialanalysis of hot spot and cold spot frequency.

1. Hot AID

Optimization of the AID sequence to make the sequence more susceptibleto somatic hypermutation resulted in an increase of about 200% in numberof hot spots (an increase of 43), and reduced the number of cold spotsby about 30% (a decrease of 23). Overall the frequency of hot spotsincreased to an average density of about 14 hot spots per 100nucleotides from an initial density of about 7 hot spots per 100nucleotides, and the overall frequency of cold spots decreased fromabout 13 cold spots per 100 nucleotides in the native gene to about 9cold spots per 100 nucleotides in the SHM susceptible form (see FIG.13).

2. Cold AID

Optimization of the canine AID sequence to make the sequence moreresistant to somatic hypermutation resulted in an increase of 186% innumber of cold spots (an increase of 68), and reduced the number of hotspots by about 35% (a decrease of 14). Overall the frequency of coldspots increased to an average density of about 25 cold spots per 100nucleotides from an initial density of about 13 cold spots per 100nucleotides, and the overall frequency of hot spots decreased from about7 hot spots per 100 nucleotides, in the native gene to about 5 hot spotsper 100 nucleotides in the SHM resistant form (see FIG. 14).

B. Cloning and Analysis

After final review to ensure that the synthetic polynucleotide sequenceis free of extraneous restriction sites, the complete polynucleotidesequence was synthesized (DNA 2.0, Menlo Park, Calif.), cloned into oneof DNA2.0's cloning vectors (see Table 10 in Example 1), sequenced toconfirm correct synthesis and tested for activity as described below andin Example 1.

To determine canine AID activity, the cold or wild type versions of AIDare co transfected with expression vectors expressing the GFP* constructthat contains a stop codon within it's coding region (as described inExample 1). Either in the presence or absence of Ig enhancer elementswithin the target vector sequence. Mutation of the stop codon by AIDresults in the creation of a functional fluorescent protein that is adirect indicator of AID activity.

In this experiment, cells are harvested by trypsinization, washed twicein PBS containing 1% w/v BSA, and resuspended in 2001 PBS/i % BSAcontaining 2 ng/ml DAPI. Cells were analyzed in the Cytopeia Influx with200 mW 488 nm and 50 mW 403 nm laser excitation. Up to one million cellsper sample were acquired and revertants were determined as percentage ofDAPI excluding live cells with detectable GFP fluorescence abovecellular background.

FIG. 16A shows the predicted effect of AID activity on protein function,in this type of assay. Of note is the observation that mutagenesis canproduce mutations that both initially restore or improve function andlater reduce or eliminate function. The balance in these two ratesgenerates early and rare mutation events that restore function, followedby secondary and tertiary mutation events that destroy function in theseproteins. The net effect of these competing rates on the observation ofgain-of-function events in a population can be seen in FIG. 16A. Giventhree different assumptions regarding number of inactivating mutationsneeded to silence GFP, one would expect to observe three very differentprofiles of reversion events as a function of time, dependent on therate of enzymatic activity of the AID.

Thus, although initial reversion rates can provide an accurateassessment of AID activity, long term studies of activity require ananalysis of the rate of extinction of activity, rather than reversion offluorescence.

To test this possibility, a cell line that is stably expressing afluorescent protein is transfected with 2 concentrations of expressionvector containing cold canine AID. Cells are stably maintained inculture and sample assayed for total fluorescence after the indicatedperiods of time.

Prior to FACS analysis, cells are harvested by trypsinization, washedtwice in PBS containing 1% w/v BSA, and resuspended in 200 μl PBS/1% BSAcontaining 2 ng/ml DAPI. Cells are analyzed in the Cytopeia Influx with200 mW 488 nm and 50 mW 403 nm laser excitation. DAPI fluorescence ismeasured through a 460/50 bandpass filter. GFP fluorescence is measuredthrough a 528/38 bandpass filter. Percent GFP expression is reported aspercentage of DAPI excluding live cells with no detectable GFPfluorescence above cellular background.

The results, shown in FIG. 16B, show a steady and sustained progressive,dose dependent decrease in GFP expression (shown as increasing GFPextinction) with time when co-expressed with increasing amounts of coldAID. The data are consistent with the hypothesis that cold AID is ableto introduce multiple mutations into a target gene, and is bothfunctional and stable when expressed in a “cold form” for many days.

To directly compare the ability of cold canine AID to exert mutagenesis,initial reversion assays are set up comparing cold canine AID with wildtype human AID. Hek 293 cells are transfected with the expressionvectors (as described above in Example 1) containing either the GFP* asdescribed above, or GFP* with the Kappa E3 and intronic enhancesinserted 5′ to the CMV promoter, together with either human or coldcanine AID. Selection for stable expression began 3 days posttransfection. Prior to FACS analysis, cells are harvested bytrypsinization, washed twice in PBS containing 1% w/v BSA, andresuspended in 200 μl PBS/1% BSA containing 2 ng/ml DAPI. Cells areanalyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW 403 nmlaser excitation. Up to one million cells per sample are acquired. DAPIfluorescence was measured through a 460/50 bandpass filter. GFPfluorescence is measured through a 528/38 bandpass filter. Percent GFPexpression is reported as percentage of DAPI excluding live cells withno detectable GFP fluorescence above cellular background.

The results show (FIG. 16C) that canine AID exhibited significantlyenhanced reversion activity compared to human AID. Also in thisexperiment is shown the effect of the kappa 3′E and intronic enhancerson the rate of reversion experienced by the target gene when these wereincluded in the expression vector. As shown inclusion of the enhancerelements further enhanced reversion frequency.

Example 3 Vectors for Somatic Hypermutation

Vectors are constructed from sub-fragments that are each synthesized byDNA2.0 (Menlo Park, Calif.). Vectors are able to simultaneously expressmultiple open reading frames and are capable of stable, episomalreplication in mammalian cells that are naturally permissive or renderedto be permissive (i.e., via co-expression of human EBP2 (Habel et al.,2004; Kapoor et al., 2001) for replication of Epstein Barr Virus (EBV)origin of replication (oriP) containing vectors.

Plasmids are rendered highly modular through the strategic placement ofone or more restriction endonuclease recognition sequences (restrictionsites) between discreet fragments throughout the vector.

A. Vectors Formats.

In the first format (FIG. 17A); vectors contain an internal ribosomeentry site (IRES) from the encephalomyocarditis virus (EMCV). Elementscontained within the vectors are operably linked together as shown inFIG. 17A and, in some cases, include the following functional elements(numbers refer to corresponding sequence information found further belowin this section): 1) CMV promoter; 2) Multicloning sites; 3) Gene ofinterest; 4) IRES; 5) Eukarybtic selectable marker such as blasticidin Sdeaminase (bsd), hygromycin phosphotransferase (hyg) orpuromycin-N-acetyl-transferase; 6) Terminator sequences, (3′untranslated region, small intron and polyA signals from SV40 (“IVSpA”)); 7) Epstein Barr Virus (EBV) origin of replication (oriP)(preceded by optional intergenic spacer region); 8) Prokaryotic originof replication Co1E1; 9) Prokaryotic selectable marker such as betalactamase (bla) gene or kanamycin (kan); 10) gene fragment for copynumber determination (such as beta actin or glucose-6-phosphatedehydrogenase (G6PDH), and Ig enhancers.

In a second format, (FIG. 17B), the expression vectors are made withoutan IRES, but contain instead an independent expression cassette forexpressing a selectable marker gene. This expression cassette caninclude, 11) the SV40 immediate early promoter (pSV) and eukaryoticselectable marker, and IVS pA as described above. Elements containedwithin the vectors are operably linked together as shown in FIG. 17 andtypically include the following functional elements: CMV promoter,multicloning sites, gene of interest, IVS pA, Epstein Barr Virus (EBV)origin of replication (oriP), pSV, selectable marker, IVS pA,prokaryotic origin of replication ColE1, prokaryotic selectable markersuch as beta lactamase (bla) gene, or kanamydin (kan), gene fragment forcopy number determination, Ig enhancers, and multicloning sites.

In a third format, (FIG. 18A) vectors contain a bidirectional promoterthat drives expression of 2 different genes oriented in oppositedirections. This vector also contains IRES sequences to generate 1 or 2bi- or tri-cistronic messages. Elements contained within the vectors areoperably linked together as shown in FIG. 18 using the same functionalelements as described previously.

In a fourth format, (FIG. 18B) vectors contain a bidirectional promoter,one or more IRES sequences that express bi- or tri-cistronic messages,and an independent, cis-linked cassette from which a eukaryoticselectable marker is expressed.

Any of the vectors can be interchanged with each other to form hybrids.In addition, any of the strong constitutive eukaryotic promoterscontained on the episomal vector can be substituted with an induciblepromoter (i.e. the reverse tetracycline transactivator promoter system[prtTA]) to achieve conditional expression of a desired gene. In thiscase, one of the other genes of interest should encode thetransactivating protein, which can be expressed in cis on the sameepisome (as shown in FIG. 19), or supplied in trans on a second,transfected episomal vector.

The orientations for the prokaryotic selectable marker and colE1 originof replication provided in sections 8 and 9 below (SEQ ID NOS: 313, 314and 315), and in FIGS. 17-19 are not absolute and can be reversed withrespect to the remainder of the vector. Similarly, the orientation ofthe independent expression cassette (pSV-selectable marker (or othergene of interest)-IVS pA) can also be reversed with respect to theremainder of the vector (i.e. transcribing toward the orip instead ofthe current portrayal of transcription away from the orip).Additionally, enhancer elements, such as Ig enhancers may be placedeither 5′ or 3′ to the gene of interest, or may excluded.

B. Representative Sequences of Functional Elements

1. A strong transcriptional promoter that works in eukaryotic cells. InFIGS. 17-19, the CMV promoter is used and the sequence is provided asSEQ ID NO: 304 (the TATA box sequence is shown underlined). The CMVpromoter is altered to remove SacI and BsrGI sites.

(SEQ ID NO: 304) AAGCTTGGCCCATTGCATSACGTTGTATCCATATCATAATATCTACATTTATATTGGCCTCATGTCCAACATTACCGCCATGTTGACATTGATTATTGACTAGTTATTAATAGTAATCAATTACGGGGTCATTAGTTCATAGCCCATATATATGGAGTTCCGCGTTACATAACTTACGGTAAATGGCCCGCCTGGCTGACCGCCCAACGACCCCCGCCCATTGACGTCAATAATGACGTATGTTCCCATAGTAACGCCAATAGGGACTTTCCATTGACGTCAATGGGTGGAGTATTTACGGTAAACTGCCCACTTGGCAGTACATCAAGTGTATCATATGCCAAGTACGCCCCCTATTGACGTCAATGACGGTAAATGGCCCGCCTGGCATTATGCCCAGTACATGACCTTATGGGACTTTCCTACTTGGCAGTACATCTACGTATTAGTCATCGCTATTACCATGGTGATGCGGTTTTGGCAGTACATCAATGGGCGTGGATAGCGGTTTGACTCACGGGGATTTCCAAGTCTCCACCCCATTGACGTCAATGGGAGTTTGTTTTGGCACCAAAATCAACGGGACTTTCCAAAATGTCGTAACAACTCCGCCCCATTGACGCAAATGGGCGGTAGGCGTGTACGGTGGGAGGTCTATATAAGCAGAGCTGGTTTAGTGAACCGTCAGATCGCCTA.

2. A region encoding multiple restriction sites termed a multicloningsite (mcs) region:

(SEQ ID NO: 305) TTCCCTGCAGGATTGTTTAAACACCAGATCTGCTTGAATCCGCGGATAAGAGGACTAGTATTCGTCTCACTAGGGAGAGCTCCTA.

3. A gene of interest such as, for example, specific binding member,antibody or fragment thereof, antibody heavy or light chain, enzyme,receptor, peptide growth hormone or transcription factor.

4. An internal ribosome entry site (IRES), in FIGS. 17-19 from theencephalomyocarditis virus (EMCV)-permits the concomitant bicistronicexpression of two open reading frames (ORF's): one 5′ to itself, and asecond 3′ to itself. A region containing 2 restriction sites (BsrGI andAscI) is shown 5′ to the IRES (lower case letters). The 3′end of theIRES includes an NgoMIV site.

(SEQ ID NO: 306) tgtacaatccgcgtgagacgatcggcgcgccCGCCCCTCTCCCTCCCCCCCCCCTAACGTTACTGGCCGAAGCCGCTTGGAATAAGGCCGGTGTGCGTTTGTCTATATGTTATTTTCCACCATATTGCCGTCTTTTGGCAATGTGAGGGCCCGGAAAGCTGGCCCTGTCTTCTTGACGAGCATTCCTAGGGGTCTTTCCCCTCTCGCCAAAGGAATGCAAGGTCTGTTGAATGTCGTGAAGGAAGCAGTTCCTCTGGAAGCTTCTTGAAGACAAACAACGTCTGTAGCGACCCTTTGCAGGCAGCGGAACCCCCCACCTGGCGACAGGTGCCTCTGCGGCCAAAAGCCACGTGTATAAGATACACCTGCAAAGGCGGCACAACCCCAGTGCCACGTTGTGAGTTGGATAGTTGTGGAAAGAGTCAAATGGCTCTCCTCAAGCGTATTCAACAAGGGGCTGAAGGATGCCCAGAAGGTACCCCATTGTATGGGATCTGATCTGGGGCCTCGGTGCACATGCTTTACATGTGTTTAGTCGAGGTTAAAAAAACGTCTAGGCCCCGCGAACCACGGGGACGTGGTTTTCCTTTGAAAAACACG ATGATAATATGGCCGGC.

5. The open reading frame (ORF) for a mammalian selectable marker gene,such as, for example, blasticidin S deaminase (bsd) (SEQ ID NO: 308),hygromycin phosphotransferase (hyg) (SEQ ID NO: 309), orpuromycin-N-acetyl-transferase (SEQ ID NO: 310). Start and stop codonsare underlined. 3′ to each ORF is an XbaI site (TCTAGA; SEQ ID NO: 307)used in the cloning step.

Blasticidin S Deaminase (bsd; Cold Spot Optimized)

(SEQ ID NO: 308) ATGGCCAAGCCTTTGTCTGAAGAAGAATCCACCCTCATTGAAAGGGCCACTGCTACAATCAACAGCATCCCCATCTCTGAAGACTACTCTGTCGCCAGCGCAGCTCTCTCCTCTGACGGGAGAATCTTCACTGGTGTCAATGTATATCATTTTACTGGGGGACCTTGCGCAGAGCTTGTGGTCCTGGGGACTGCTGCTGCTGCTGCAGCCGGAAACCTGACTTGTATCGTCGCCATAGGGAATGAGAACAGAGGCATCTTGAGCCCCTGTGGGAGATGCAGACAAGTCCTCCTGGACCTCCATCCTGGGATCAAAGCGATAGTGAAGGACAGTGATGGACAGGCCACAGCCGTTGGGATCAGGGAGTTGCTGCCATCTGGTTATGTGTGGGAGGGCTAAT CTAGA.

Hygromycin Phosphotransferase (hyg; Cold Spot Optimized)

(SEQ ID NO: 309) ATGAAAAAGCCTGAACTGACTGCGACCTCTGTTGAGAAGTTTTTAATAGAGAAGTTTGACTCTGTGTGAGACCTCATGCAGCTTTCTGAGGGAGAGGAGTCTAGAGCCTTTAGCTTTGATGTGGGGGGGAGAGGCTATGTCCTGAGAGTCAATAGCTGTGCAGATGGTTTCTACAAAGATAGGTATGTCTATAGACATTTTGCATCCGCCGCCCTCCCCATTCCAGAGGTCCTTGACATTGGGGAATTCTCAGAGAGCCTGACCTATTGCATTTCCCGGAGAGCCCAGGGTGTGACTCTTCAAGACCTGCCTGAGACAGAACTCCCTGCAGTGGTCCAGCCCGTCGCCGAGGCCATGGATGCAATCGCCGCCGCAGACCTGAGCCAGACCTCGGGGTTTGGCCCCTTTGGCCCCCAGGGGATAGGCCAATACACTACATGGAGAGATTTCATATGCGCTATTGCTGACCCCCATGTGTATCACTGGCAAACTGTGATGGACGACACAGTCTCAGCCTCTGTCGCACAAGCGCTGGACGAGCTGATGCTTTGGGCCGAGGACTGCCCAGAGGTCAGACATCTCGTCCATGCCGACTTTGGGTCAAACAATGTCCTGACGGACAATGGGAGAATCAGTGCTGTGATTGACTGGAGCGAGGCCATGTTTGGGGACTCCCAATACGAGGTCGCCAACATCTTCTTCTGGAGACCCTGGTTGGCTTGTATGGAGCAGCAGACCCGTTACTTTGAGAGGAGGCATCCAGAGCTCGCTGGGAGCCCTAGATTGAGGGCCTATATGCTCAGGATAGGGCTTGACCAACTCTATCAGAGCTTGGTTGACGGCAATTTTGATGACGCAGCTTGGGCTCAGGGGAGATGCGACGCCATAGTGAGGAGTGGGGCCGGGACTGTCGGGAGAACTCAGATCGCCAGGAGGTCAGCTGCCGTCTGGACTGACGGCTGTGTAGAAGTCTTAGCCGACTCTGGGAACAGGAGACCCAGCACTCGTCGAGAGGCCAAGGAATGATCTAGA.

Puromycin-N-Acetyl-Transferase (Pur; Wild Type Sequence).

Contains a Kozak consensus sequence immediately 5′ to the start codon(underlined). Stop codon is also underlined.

(SEQ ID NO: 310) CACCATGACCGAGTACAAGCCGACGGTGCGCCTCGCCACCCGCGACGACGTCCCCCGGGCCGTTCGCACCCTCGCCGCCGCGTTCGCCGACTACCCCGCCACGCGCCACACCGTGGACCCGGACAGGCACATCGAGCGGGTCACCGAGCTGCAAGAACTCTTCCTCACGCGCGTCGGGCTCGACATCGGCAAGGTGTGGGTCGCGGACGACGGCGCCGCTGTGGCGGTCTGGACCACGCCGGAGAGCGTCGAAGCGGGGGCGGTGTTCGCCGAGATCGGCCCGCGCATGGCCGAGTTGAGCGGTTCCCGGCTGGCCGCGCAGCAAGAGATGGAAGGCCTCCTGGCGCCGCACCGGCCCAAGGAGCCCGCGTGGTTCCTGGCTACCGTCGGAGTCTCGCCCGACCACCAGGGCAAGGGTCTGGGCAGCGCCGTCGTGCTCCCCGGAGTGGAGGCTGCCGAGCGTGCCGGGGTGCCCGCCTTCCTCGAGACCTCCGCGCCCCGCAACCTCCCCTTCTACGAGCGGCTCGGCTTCACCGTCACCGCCGACGTCGACGTGCCCGAAGGACCGCGCACCTGGTGCATGACCCGCAAGCCCGGTGC CTGATCTAGA.

6. Terminator Sequences, IVS-pA (Shown with 3′ BamH I).

(SEQ ID NO: 311) GGATCTTTGTGAAGGAACCTTACTTCTGTGGTGTGACATAATTGGACAAACTACCTACAGAGATTTAAAGCTCTAAGGTAAATATAAAATTTTTAAGTGTATAATGTGTTAAACTACTGATTCTAATTGTTGTGGTATTTTAGATTCCAACCTATGGAACTTATGAATGGGAGCAGTGGTGGAATGCCTTTAATGAGGAAAAGCTGTTTTGCTCAGAAGAAATGCCATCTAGTGATGATGAGGCTACTGCTGACTCTCAACATTCTACTCCTCCAAAAAAGAAGAGAAAGGTAGAAGACCCCAAGGACTTTCCTTCAGAATTGGTAAGTTTTTTGAGTCATGCTGTGTTTAGTAATAGAACTCTTGCTTGCTTTGCTATTTACACCACAAAGGAAAAAGCTGCACTGCTATACAAGAAAATTATGGAAAAATATTTGATGTATAGTGCCTTGACTAGAGATCATAATCAGCCATACCACATTTGTAGAGGTTTTACTTGCTTTAAAAAACCTCCCACACCTCCCCCTGAACCTGAAACATAAAATGAATGCAATTGTTGTTGTTAACTTGTTTATTGCAGCTTATAATGGTTACAAATAAAGCAATAGCATCACAAATTTCACAAATAAAGCATTTTTATCACTGCATTCTAGTTGTGGTTTGTCCAAACTCATCAATGTATCTTATCATGTCTGGATC C.

Sequence of EBV oriP. This element permits episomal replication in EBVoriP permissive cells that express Epstein Barr Nuclear Antigen 1(EBNA1). The orip sequence is preceded by an optional intergenic spacerregion (small letters):

(SEQ ID NO: 312) actgtcttctttatcatgcaactcgtaggacaggtgccctggccgggtccGCAGGAAAAGGACAAGCAGCGAAAATTCACGCCCCCTTGGGAGGTGGCGGCATATGCAAAGGATAGCACTCCCACTCTACTACTGGGTATCATATGCTGACTGTATATGCATGAGGATAGCATATGCTACCCGGATACAGATTAGGATAGCATATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGATAGCCTATGCTACCCAGATATAAATTAGGATAGCATATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGATAGCCTATGCTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGATAGCATATGCTATCCAGATATTTGGGTAGTATATGCTACCCAGATATAAATTAGGATAGCATATACTACCCTAATGTCTATTAGGATAGCATATGCTACCCGGATACAGATTAGGATAGCATATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGATAGCCTATGCTACCCAGATATAAATTAGGATAGCATATACTACCCAGATATAGATTAGGATAGCATATGCTACCCAGATATAGATTAGGATAGCCTATGCTACCCAGATATAGATTAGGATAGCATATGCTATCCAGATATTTGGGTAGTATATGCTACCCATGGCAACATTAGCCCACCGTGCTCTCAGCGACCTCGTGAATATGAGGACCAACAACCCTGTGCTTGGCGCTCAGGCGCAAGTGTGTGTAATTTGTCCTCCAGATCGCAGCAATCGCGCCCCTATCTTGGCCCGCCCACCTACTTATGCAGGTATTCCCCGGGGTGCCATTAGTGGTTTTGTGGGCAAGTGGTTTGACCGCAGTGGTTAGCGGGGTCAGAATCAGCCAAGTTATTACACCCTTATTTTACAGTCCAAAACCGCAGGGCGGCGTGTGGGGGCTGACGCGTGCCATCACTCCACAATTTCAAGAGAAAGAGTGGCCACTTGTCTTTGTTTATGGGCCCCATTGGCGTGGAGCCCCGTTTAATTTTCGGGGGTGTTAGAGACAACCAGTGGAGTCCGCTGCTGTCGGCGTCCACTCTTTCCCCTTGTTACAAATAGAGTGTAACAACATGGTTCACCTGTCTTGGTCCCTGCCTGGGACACATCTTAATAACCCCAGTATCATATTGCACTAGGATTATGTGTTGCCCATAGCCATAAATTCGTGTGAGATGGACATCCAGTCTTTACGGCTTGTCCCCACCCCATGGATTTCTATTGTTAAAGATATTCAGAATGTTTCATTCCTACACTAGGATTTATTGCCCAAGGGGTTTGTGAGGGTTATATTGGTGTCATAGCACAATGCCACCACTGAACCCATCGTCCAAATTTTATTCTGGATGCGTCACCTGAAACCTTGTTTTCGAGCACCTCACATACACCTTACTGTTCACAACTCAGCAGTTATTCTATTAGCTAAACGAAGGAGAATGAAGAAGCAGGCGAAGATTCAGGAGAGTTCACTGCCCGCTCCTTGATCTTCAGCCACTGCCCTTGTGACTAAAATGGTTCACTACCCTCGTGGAATCCTGACCCCATGTAAATAAAACCGTGACAGCTCATGGGGTGGGAGATATCGCTGTTCCTTAGGACCCTTTTACTAACCCTAATTCGATAGCATATGCTTCCCGTTGGGTAACATATGCTATTGAATTAGGGTTAGTCTGGATAGTATATACTACTACCCGGGAAGCATATGCTACCCGTTTAGGGTTAACAAGGGGGCCTTATAAACACTATTGCTAATGCCCTCTTGAGGGTCCGCTTATCGGTAGCTACACAGGCCCCTCTGATTGACGTTGGTGTAGCCTCCCGTAGTCTTCCTGGGCCCCTGGGAGGTACATGTCCCCCAGCATTGGTGTAAGAGCTTCAGCCAAGAGTTACACATAAAGG.

8. Sequence of Escherichia coli origin of replication colEI, derivedfrom vector pJ15 and pJ31 from DNA2.0 (Menlo Park, Calif.): colE1

(SEQ ID NO: 313) AAAAGGGGCCCGAGCTTAAGACTGGCCGTCGTTTTACAACACAGAAAGAGTTTGTAGAAACGCAAAAAGGCCATCCGTCAGGGGCCTTCTGCTTAGTTTGATGCCTGGCAGTTCCCTACTCTCGCCTTCCGCTTCCTCGCTCACTGACTCGCTGCGCTCGGTCGTTCGGCTGCGGCGAGCGGTATCAGCTCACTCAAAGGCGGTAATACGGTTATCCACAGAATCAGCGCATAACGCAGGAAAGAACATGTGAGCAAAAGGCCAGCAAAAGGCCAGGAACCGTAAAAAGGCCGCGTTGCTGGCGTTTTTCCATAGGCTCCGCCCCCCTGACGAGCATCACAAAAATCGACGCTCAAGTCAGAGGTGGCGAAACCCGACAGGACTATAAAGATACCAGGCGTTTCCCCCTGGAAGCTCCCTCGTGCGCTCTCCTGTTCCGACCCTGCCGCTTACCGGATACCTGTCCGCCTTTCTCCCTTCGGGAAGCGTGGCGCTTTCTCATAGCTCACGCTGTAGGTATCTCAGTTCGGTGTAGGTCGTTCGCTCCAAGCTGGGCTGTGTGCACGAACCCCCCGTTCAGCCCGACCGCTGCGCCTTATCCGGTAACTATCGTCTTGAGTCCAACCCGGTAAGACACGACTTATCGCCACTGGCAGCAGCCACTGGTAACAGGATTAGCAGAGCGAGGTATGTAGGCGGTGCTACAGAGTTCTTGAAGTGGTGGGCTAACTACGGCTACACTAGAAGAACAGTATTTGGTATCTGCGCTCTGCTGAAGCCAGTTACCTTCGGAAAAAGAGTTGGTAGCTCTTGATCCGGCAAACAAACCACCGCTGGTAGCGGTGGTTTTTTTGTTTGCAAGCAGCAGATTACGCGCAGAAAAAAAGGATCTCAAGAAGATCCTTTGATCTTTTCTACGGGGTCTGACGCTCAGTGGAACGACGCGCGCGTAACTCACGTTAAGGGATTTTGGTCATGAGCTTGCGCCGTCCCGTCAAGT CAGCGTAATGCTCTG.

9A. Sequence of beta lactamase (bla) gene for resistance. The openreading frame (ORF) is shown in reverse orientation.

(SEQ ID NO: 314) CTTACCAATGCTTAATCAGTGAGGCACCTATCTCAGCGATCTGTCTATTTCGTTCATCCATAGTTGCCTGACTCCCCGTCGTGTAGATAACTACGATACGGGAGGGCTTACCATCTGGCCCCAGCGCTGCGATGATACCGCGAGAACCACGCTCACCGGCTCCGGATTTATCAGCAATAAACCAGCCAGCCGGAAGGGCCGAGCGCAGAAGTGGTCCTGCAACTTTATCCGCCTCCATCCAGTCTATTAATTGTTGCCGGGAAGCTAGAGTAAGTAGTTCGCCAGTTAATAGTTGCGCAACGTTGTTGCCATCGCTAGAGGCATCGTGGTGTCACGCTCGTCGTTTGGTATGGCTTCATTCAGCTCCGGTTCCCAACGATCAAGGCGAGTTACATGATCCCCCATGTTGTGCAAAAAAGCGGTTAGCTCCTTCGGTCCTCCGATCGTTGTCAGAAGTAAGTTGGCCGCAGTGTTATCACTCATGGTTATGGCAGCACTGCATAATTCTCTTACTGTCATGCCATCCGTAAGATGCTTTTCTGTGACTGGTGAGTACTCAACCAAGTCATTCTGAGAATAGTGTATGCGGCGACCGAGTTGCTCTTGCCCGGCGTGAATACGGGATAATACCGCGCCACATAGCAGAACTTTAAAAGTGCTCATCATTGGAAAACGTTCTTCGGGGCGAAAACTCTCAAGGATCTTACCGCTGTTGAGATCCAGTTCGATGTAACCCACTCGTGCACCCAACTGATCTTCAGCATCTTTTACTTTCACCAGCGTTTCTGGGTGAGCAAAAACAGGAAGGCAAAATGCCGCAAAAAAGGGAATAAGGGCGACACGGAAATGTTGAATACTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGGTCATAACACCCCTTGCAGTGCGACTAACGGCATGAAGCTCGTCGGGGCGTACG.

9B. Sequence of kanamycin (kan), derived from vector pJ31 from DNA2.0(Menlo Park, Calif.). The open reading frame (ORF) is shown in reverseorientation.

(SEQ ID NO: 315) CTTAGAAAAACTCATCGAGCATCAAATGAAACTGCAATTTATTCATATCAGGATTATCAATACCATATTTTTGAAAAAGCCGTTTCTGTAATGAAGGAGAAAACTCACCGAGGCAGTTCCATAGGATGGCAAGATCCTGGTATCGGTCTGCGATTCCGACTCGTCCAACATCAATACAACCTATTATTTCCCCTCGTCAAAAATAAGGTTATCAAGTGAGAAATCACCATGAGTGACGACTGAATCCGGTGAGAATGGCAAAAGTTTATGCATTTCTTTCCAGACTTGTTCAACAGGCCAGCCATTACGCTCGTCATCAAAATCACTCGCATCAACCAAACCGTTATTCATTCGTGATTGCGCCTGAGCGAGGCGAAATACGCGATCGCTGTTAAAAGGACAATTACAAACAGGAATCGAGTGCAACCGGCGCAGGAACACTGCCAGCGCATCAACAATATTTTCACCTGAATCAGGATATTCTTCTAATACCTGGAACGCTGTTTTTCCGGGGATCGCAGTGGTGAGTAACCATGCATCATCAGGAGTACGGATAAAATGCTTGATGGTCGGAAGTGGCATAAATTTCCGTCAGCCAGTTTAGTCTGACCATCTCATCTGTAACATCATTGGCAACGCTACCTTTGCCATGTTTCAGAAACAACTCTGGCGCATCGGGCTTCCCATACAAGCGATAGATTGTCGCACCTGATTGCCCGACATTATCGCGAGCCCATTTATACCCATATAAATCAGCATCCATGTTGGAATTTAATCGCGGCCTCGACGTTTCCCGTTGAATATGGCTCATATTCTTCCTTTTTCAATATTATTGAAGCATTTATCAGGGTTATTGTCTCATGAGCGGATACATATTTGAATGTATTTAGAAAAATAAACAAATAGGGGTCAGTGTTACAACCAATTAACCAATTCTGAACATTATCGCGAGCCCATTTATACCTGAATATGGCTCATAACACCCGTTGCAGTGCGACTAAGGGCATGAAGCTCGTCGGGGAAATAATGATTTTATTTTGACTGATAGTGACCTGTTCGTTGCAACAAATTGATAAGCAATGCTTTCTTATAATGCCAACTTTGTACAAGAAAGCTGGGTTTTTTTTTTAGCCTGCTTTTTTGTACAAAGTTGGCATTATAAAAAAGCATTGCTCATCAATTTGTTGCAACGAACAGGTCACTATCAGTCAAAATAAAATCATTATTT.

10. A moiety used for determination of episomal copy number per cell.Ideally, the moiety should contain a sequence that exists uniquely inthe genome. Shown below are 2 fragments, beta actin and G6PDH that canbe used in vectors known in the art or described herein. Each fragmentis bounded by a BsiWI and a Cla I site.

beta actin moiety

(SEQ ID NO: 316) CGTACGTACTCCTGCTTGCTGATCCACATCTGCTGGAAGGTGGACAGCGAGGCCAGGATGGAGCCGCCGATCCACACGGAGTACTTGCGCTCAGGAGGAGCAATGAAGCTTATCTGAGGAGGGAAGGGGACAGGCAGTGAGGACCCTGGATGTGACAGCTCCAAGCTTCCACACACCACAGGACCCCACAGCCGACCTGCCCAGGTCAGCTCAGGCAGGAAAGACACCCACCTTGATCTTCATTGTGCTGGGTGCCAGGGCAGTGATCTCCTTCTGCATCCTGTCATCGAT.

Human Glucose-6-Phosphate Dehydrogenase (hG6PDH) Moiety

(SEQ ID NO: 317) CGTACGAGGTGAGGCTGCAGTTCCATGATGTGTCCGGCGACATCTTCCACCAGCAGTGCAAGCGCAACGAGCTGGTGATCCGCGTGCAGCCCAACGAGGCCGTGTACCAGAGAAGGAGCAGTGTGGAGGGTGGGCGGCCTGGGCCCGGGGGACTCCACATGGTGGCAGGCAGTGGCATCAGCAAGACACTCTCTCCCTCACAGAACGTGAAGCTCCCTGACGCCTACGAGCGCCTCATCCTGGACGTCTTCTGCGGGAGCCAGATGCACTTCGTGCGCAGGAATCGAT.

11. pSV, immediate early promoter from SV40. The sequence is preceded bya BstBI site and followed by an NgoMIV site.

(SEQ ID NO: 318) TTCGAAGCTGTGGAATGTGTGTCAGTTAGGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAAGCAGGTGTGGAAAGTCCCCAGGCTCCCCAGCAGGCAGAAGTATGCAAAGCATGCATCTCAATTAGTCAGCAACCATAGTCCCGCCCCTAACTCCGCCCATCCCGCCCCTAACTCCGCCCAGTTCCGCCCATTCTCCGCCCCATGGCTGACTAATTTTTTTTATTTATGCAGAGGCCGAGGCCGCCTCGGCCTCTGAGCTATTCCAGAAGTAGTGAGGAGGCTTTTTTGGAGGCCTAGGCTTTTGCAAAAAGCTCTGAGCCCTCACAAGGAGCCGGC.

Ig Enhancers

Representative Ig enhancer sequences include the heavy or light chainenhancers. The Kappa 3′ enhancer region (Ek3′) (See Meyer, K. B. andNeuberger, M. S., EMBO J. 8 (7), 1959-1964 (1989)), and Kappa intronicenhancer region, Eki LOCUS L80040 7466 bp ROD 02-SEP-2003 are shownbelow by way of example. At least 1 major active element within theenhancer regions is the E box sequence: CAGGTG(N)₁₃CAGGTG (SEQ ID NO:319) [core sequence: CANNTG; SEQ ID NO: 320] Storb et al., Immunity19:235-242, 2003). The Ek3′ and Eki enhancer elements are obtained fromDr. Neuberger (MRC, UK). The Ek3′ sequence is amplified by PCR fromNeuberger plasmid identification #1352, using the following primers,which contain an XhoI and EcoRI site, respectively, that are used forcloning:

(SEQ ID NO: 321) GACTACCTCGAGccagcttaggctacacagag andGTAGTCGAATTCCCACATGTCTTACATGGTATATG.

The Eki enhancer sequence is amplified from Dr. Neuberger's vector(identification #Me123) using oligonucleotidesGACTACGAATTCtcctgaggacacagtgatag (SEQ ID NO: 322) andGTAGTCGCGGCCGCCTAGTTCCTAGCTACTTCTTTA (SEQ ID NO: 323), which encode anEcoRI and NotI restriction site, respectively. Resulting fragments aredigested with the appropriate restriction enzyme, and clonedsequentially into mcs2 (described in below): Ek3′ is cloned into theXhoI and EcoRI sites of mcs2, and the resulting plasmid is then digestedwith EcoRI plus NotI into which the Eki fragment is subsequently ligatedto generate vector AB156.

As described above, E boxes are known to be present in the kappaenhancer region. Consequently, a synthetic cassette consisting of 3tandemly arrayed E boxes is synthesized using the complementaryoligonucleotides AATTCaggtgctggggtagggagcaggtgctacactgcagaccaggtgctGC(SEQ ID NO: 324) andggccgcagcacctggtctgcagtgtagcacctgctccctaccccagcacctg (SEQ ID NO: 325),which when annealed contain EcoRI and NotI overhangs. The annealed oligoproduct is thus cloned into the EcoRI and NotI sites of mcs2 to generatevector AB157.

A representative Ig-kappa locus 3′ enhancer element is listed below.(Accession number X15878)

(SEQ ID NO: 326) CCAGCTTAGGCTACACAGAGAAACTATCTAAAAAATAATTACTAACTACTTAATAGGAGATTGGATGTTAAGATCTGGTCACTAAGAGGCAGAATTGAGATTCGAAGCCAGTATTTTCTACCTGGTATGTTTTAAATTGCAGTAAGGATCTAAGTGTAGATATATAATAATAAGATTCTATTGATCTCTGCAACAACAGAGAGTGTTAGATTTGTTTGGAAAAAAATATTATCAGCCAACATCTTCTACCATTTCAGTATAGCACAGAGTACCCACCCATATCTCCCCACCCATCCCCCATACCAGACTGGTTATTGATTTTCATGGTGACTGGCCTGAGAAGATTAAAAAAAGTAATGCTACCTTATTGGGAGTGTCCCATGGACCAAGATAGCAAGTGTCATAGCTACCGTCACACTGCTTTGATCAAGAAGACCCTTTGAGGAACTGAAAACAGAACCTTAGGCACATCTGTTGCTTTCGCTCCCATCCTCCTCCAACAGCCTGGGTGGTGCACTCCACACCCTTTCAAGTTTCCAAAGCCTCATACACCTGCTCCCTACCCCAGCACCTGGCCAAGGCTGTATCCAGCACTGGGATGAAAATGATACCCCACCTCCATCTTGTTTGATATTACTCTATCTCAAGCCCCAGGTTAGTCCCCAGTCCCAATGCTTTTGCACAGTCAAAACTCAACTTGGAATAATCAGTATCCTTGAAGAGTTCTGATATGGTCACTGGGCCCATATA CCATGTAAGACATGTGG.

A representative Kappa intronic enhancer region, Eki is presented below:

(SEQ ID NO: 327) TCGTGAGGACACAGTGATAGGAACAGAGCCACTAATCTGAAGAGAACAGAGATGTGACAGACTACACTAATGTGAGAAAAACAAGGAAAGGGTGACTTATTGGAGATTTCAGAAATAAAATGCATTTATTATTATATTCCCTTATTTTAATTTTCTATTAGGGAATTAGAAAGGGCATAAAGTGCTTTATCCAGTGTTATATTAAAAGCTTAATGTATATAATCTTTTAGAGGTAAAATCTACAGCCAGCAAAAGTCATGGTAAATATTCTTTGACTGAACTCTCACTAAACTCCTCTAAATTATATGTCATATTAACTGGTTAAATTAATATAAATTTGTGACATGACCTTAACTGGTTAGGTAGGATATTTTTCTTCATGCAAAAATATGACTAATAATAATTTAGCACAAAAATATTTCCCAATACTTTAATTCTGTGATAGAAAAATGTTTAACTCAGCTAGTATAATCCCATAATTTTGAAAACTATTTATTAGCTTTTGTGTTTGACCCTTCCCTAGCCAAAGGCAACTATTTAAGGACCCTTTAAAACTCTTGAAACTACTTTAGAGTCATTAAGTTATTTAACCACTTTTAATTACTTTAAAATGATGTCAATTCCCTTTTAACTATTAATTTATTTTAAGGGGGGAAAGGCTGCTCATAATTCTATTGTTTTTCTTGGTAAAGAACTCTCAGTTTTCGTTTTTACTACCTCTGTCACCCAAGAGTTGGCATCTCAACAGAGGGGACTTTCCGAGAGGCCATCTGGCAGTTGCTTAAGATCAGAAGTGAAGTCTGCCAGTTCCTCCCAGGCAGGTGGCCCAGATTACAGTTGACCTGTTCTGGTGTGGCTAAAAATTGTCCCATGTGGTTACAAACCATTAGACCAGGGTCTGATGAATTGCTCAGAATATTTCTGGACACCCAAATACAGACCCTGGCTTAAGGCCCTGTCCATACAGTAGGTTTAGCTTGGCTACACCAAAGGAAGCCATACAGAGGCTAATACCAGAGTATTCTTGGAAGAGACAGGAGAAATGAAAGCCAGTTTCTGCTCTTACCTTATGTGCTTGTGTTCAGACTCCCAAACATCAGGAGTGTCAGATAAACTGGTCTGAATCTCTGTCTGAAGCATGGAACTGAAAAGAATGTAGTTTCAGGGAAGAAAGGCAATAGAAGGAAGCCTGAGAATATCTTCAAAGGGTCAGACTCAATTTACTTTCTAAAGAAGTAGCTAGGAACTA G.

Vector construction is described in priority U.S. application No.60/902,414.

Example 4 Identification of Representative Human Scaffold AntibodyVariable Domains

To identify the germline variable antibody domains that are used mostoften in the generation of mature antibodies during the process ofrecombination, SHM, and selection, 850 antibody heavy and light chainsequences available from the PDB database to the 39× light chains, 44 κlight chains, and 55 heavy chains germline variable domain sequences arecompared.

In addition, a similar comparison is made to 21,000 Genbank Human(www.ncbi.nih.gov/genbank) IgG heavy and light chain sequences. Usingthe PDB database as a source for comparison has several advantages: itcontains antibodies bound almost entirely to peptides and proteins, manyto proteins of therapeutic interest, most of the bound antibodies bindwith high-affinity to their targets, and antibody sequences are derivedfrom many sources and libraries. Mapping variable domains to Genbanksequences provides a statistically significant analysis of the commonlyused germline sequence.

Variable domain template identification is conducted by creatingcladograms for each of the three variable domain isoform classes usingthe phylip 3.65 ProtMLK program (“Numerical methods for inferringevolutionary trees.” Quarterly Review of Biology 57:379-404). Thisprogram implements the maximum likelihood method for protein amino acidsequences under the constraint that the trees estimated must beconsistent with a molecular clock, the assumption that the tips of thetree are all equidistant, in branch length, from its root. It uses theDayhoff probability model of change between amino acids with thefollowing assumptions: a) each position in the sequence evolvesindependently; b) different lineages evolve independently; c) eachposition undergoes substitution at an expected rate which is chosen froma series of specified rates (each with a probability of occurrence); d)all relevant positions are included in the sequence, not just those thathave changed or those that are “phylogenetically informative”; and e)the probabilities of change between amino acids are given by the modelof Jones, Taylor, and Thornton (1992), the PMB model of Veerassamy,Smith and Tillier (2003), or the PAM model of Dayhoff (Dayhoff and Eck,1968; Dayhoff et. al., 1979).

In addition, each of the germline variable domains are evaluated todetermine how frequently each germline variable domain was the likelyantecedent for a mature antibody observed in a sequence or structuraldatabase. Presumably, each of these variable domains contributesdifferentially to the binding distinct antigen classes (proteins,haptens, polysaccharides, etc). Understanding which variable sequencescontribute commonly to binding proteins targets and incorporating thesevariable template regions provides for the creation of a functionallyenriched antibody library.

This comparison demonstrated that the variable regions for the λ lightchains, κ light chains, and heavy chain isoforms segregate into a smallnumber of highly related sub-clades. It is observed (FIGS. 20 (A), (B)and (C),) that certain members of these sub-clades contribute many timesto antibodies found in the PDB and Genbank databases, whereas othergermline variable regions are seldom observed to contribute. Forinstance, variable domains IGLV4-IGLV11 are not observed to contributeto antibody sequences from the PDB and rarely in Genbank, whereas usageof IGLV1-IGLV3 variable domains account for almost all mature antibodysequences containing a IGL light chain.

Eighteen germline variable sequences are identified that represent mostλ, κ, and H sub-clades and that are used often in the generation ofmature antibodies during the process of recombination, SHM, selection.In FIG. 20, the heavy chain, κ light chain, and λ light chain isoformvariable domain cladograms and frequency distributions for germlineusage are shown, with an @ highlighting those members chosen for use asa variable region template for the semi-synthetic antibody librariesdescribed herein. Table 11 lists the selected template variable regionsthat are identified for synthesis as described below. While we haveselected a set of highly used and represented variable template regionsfor constructing the library, the minor differences between members ofdifferent variable regions and the ability of antibodies to employdifferent variable regions to recognize the same epitope, suggest thatone might also use other germline variable regions, subsets of thoseregions chosen in Table 11, or some combination of both as templates forthe antibody library described herein.

For example, Heavy Chains IGHV4-55, IGHV4-61, IGHV2-5, IGHV3-30,IGHV3-74, IGHV3-72, IGHV3-66, IGHV3-53, IGHV146 and IGHV7.4-1; KappaLight Chains IGKV2.24, IGKV2D-30, IGKV2.29, IGKV2.28, IGKV7-3,IGKV1D-33, IGKV1-9 and IGKV6D41; and Lambda Light Chains IGLV4-69,IGLV6-57, IGLV141, IGLV1-47, IGLV2-23, IGLV3-1 and IGLV3-10.

TABLE 11 Template Variable Regions Identified for the Semi-SyntheticAntibody Libraries Heavy Chains Kappa Light Chains Lambda Light ChainsIsoform IGHV Isoform IGKV Isoform IGLV Most preferred IGHV6-1 IGKV4-1IGLV2-11 IGHV3-30 IGKV3-20 IGLV1-40 IGHV4-34 IGKV2D-30 IGLV3-21 IGHV3-7IGKV1D-39 IGLV7-43 IGHV4-59 IGKV1-33 IGHV3-23 IGHV5-51 IGHV1-69 IGHV1-2

Example 5 Synthesis and Cloning of Human Scaffold Antibody VariableDomains

The amino acid sequences and NCBI Entrez Gene IDs of the 9 variableregion scaffolds chosen for use in the construction of the initial heavychain library repertoire, the 5 variable region scaffolds chosen for usein the construction of kappa light chain library repertoire, and the 4variable region scaffolds chosen for use in the construction of thelambda light chain library repertoire are shown in Table 12, below. Thegene identifier Entrez Gene ID can be found atwww.ncbi.nln.nih.gov/entrez/query.fcgi?CMD=search&DB=gene.

TABLE 12 Variable Region Scaffolds NCBI Variable Entrez Region Gene NameID Amino acid sequence IGHV6-1 28385 MSVSFLIFLPVLGLPWGVLSQVQLQQSGPGLVKP(H1) SQTLSLTCAISGDSVSSNSAAWNWIRQSPSRGLEWLGRTYYRSKWYNDYAVSVKSRITINPDTSKNQF SLQLNSVTPEDTAVYYCAR (SEQ ID NO: 328)IGHV4-34 28395 MKHLWFFLLLVAAPRWVLSQVQLQQWGAGLLKPS (H2)ETLSLTCAVYGGSFSGYYWSWIRQPPGKGLEWIG EINHSGSTNYNPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV (SEQ ID NO: 329) IGHV4-59 28392MKLHLWFFLLLVAAPRWVLSQVQLQESGPGLVKP (H3)SETLSLTCTVSGGSISSYYWSWIRQPPGKGLEWI GYIYYSGSTNYNIPSLKSRVTISVDTSKNQFSLKLSSVTAADTAV (SEQ ID NO: 330) IGHV3-30-3 57290MEFGLSWVFLVALLRGVQCQVQLVESGGGVVQPG (H4)RSLRLSCAASGFTFSSYAMHWVRQAPGKGLEWVA VISYDGSNKYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV (SEQ ID NO: 331) IGHV3-7 28452MELGLSWVFLVAILEGVQCEVQLVESGGGLVQPG (H5)GSLRLSCAASGFTFSSYWMSWVRQALPGKGLEWV ANIKQDGSEKYYVDSVKGRFTISRDNAKNSLYLQMNSLRAEDTAV (SEQ ID NO: 332) IGHV3-23 28442MEFGLSWLFLVAKIKGVQCEVQLLESGGGLVQPG (H6)GSLRLSCAASGFTFSSYAMSWVRQAPGKGLEWVS MSGSGGSTYYADSVKGRFTISRDNSKNTLYLQMNSLRAEDTAV (SEQ ID NO: 333) IGHV5-51 28388MGSTAILALLLAVLQGVCSEVQLVQSGAEVKKPG (H7)ESLKISCKGSGYSFTSYWIGWVRQMPGKGLEWMG IIYPGDSDTRYSPSFQGQVTISADKSISTAYLQWSSLKASDTAV (SEQ ID NO: 334) IGHV1-2 28474MDWTWRILFLVAAATGAHSQVQLVQSGAEVKKPG (H8)ASVKVSCKASGYTFTGYYMHWVRQAPGQGLEWMG RINPNSGGTNYAQKFQGRVTSTRDTSISTAYMELSRLRSDDTAV (SEQ ID NO: 335) IGHV1-69 28461MDWTWRFLFVVAAATGVQSQVQLVQSGAEVKKPG (H9)SSVKVSCKASGGTFSSYAISWVRQAPGQGLEWMG GIIPIFGTANYAQKFQGRVTITADESTSTAYMELSSLRSEDTAV (SEQ ID NO: 336) IGKV2-30 28919MRLPAQLLGLLMLWVPGSSGDVVMTQSPLSLPVT (K1)LGQPASISCRSSQSLVYSDGNTYLNWFQQRPGQS PRRLIYKVSNWDSGVPDRIFSGSGSGTDFTLKISRVEAEDVAVY (SEQ ID NO: 337) IGKV4-1 28908MVLQTQVFISLLLWISGAYGDIVMTQSPDSLAVS (K2)LGERATINCKSSQSVLYSSNNKNYLAWYQQKLPG QPPLLLIYWASTRESGVPDRFSGSGSGTDFTLTISSLQAEDVAVY (SEQ ID NO: 338) IGKV1-33 28933MDMRVPAQLLGLLQLWLSGARCDIQMTQSPSSLS (K3)ASVGDRVTITCQASQDISNYLNWYQQKPGKAPKL LIYDASNLETGVPSRFSGSGSGTDFTFTISSLQPEDIAVY (SEQ ID NO: 339) IGKV1D-39 28893MDMRVPAQLLGLLLLWLRGARCDIQMTQSPSSLS (K4)EDIAVYASQSISSYLNWYQQKPGKAPKLLIYAAS SLQSGVIPSRFSGSGSGTDFTLTISSLQPEDFAV Y(SEQ ID NO: 340) IGKV3-20 28912 METPAQLLFLLLLWLPDTTGEIVLTQSPGTLSLS (K5)PGERATLSCRASQSVSSSYLAWYQQKPGQAIPRL LIYGASSRATGIPDRFSGSGSGTDFTLTISRLEPEDFAVY (SEQ ID NO: 341) IGLV7-43 28776MAWTPLFLFLLTCCPGSNSQTVVTQEPSLTVSPG (L1)GTVTLTCASSTGAVTSGYYPNWFQQKPGQAPRAL IYSTSNKHSWTPARFSGSLLGGKAALTLSGVQPEDEA (SEQ ID NO: 342) IGLV1-40 28825 MAWSPLLLTLLAHCTGSWAQSVLTQPPSVSGAPG(L2) QRVTISCTGSSSNIGAGYDVHWYQQLPGTAPKLLIYGNSNRPSGVPDRFSGSKSGTSASLAITGLQAE DEA (SEQ ID NO: 343) IGLV2-11 28816MAWSPLLLTLLAHCTGSWAQSALTQPRSVSGSPG (L3)QSVTISCTGTSSDVGGYNYVSWYQQHPGKAPKLM IYDVSKRPSGVPDRFSGSKSGNTASLTISGLQAEDEA (SEQ ID NO: 344) IGLV3-21 28796 MAWTVLLLGLLSHCTGSVTSYVLTQPPSVSVAPG(L4) KTARITCGGNNIGSKSVHWYQQKPGQAPVLVIYYDSDRPSGIPERFSGSNSGNTATLTISRVEAEDEA (SEQ ID NO: 345)

A sequence encoding the H2kk peritransmembrane, transmembrane andcytoplasmic domains was appended to the human IgG1 heavy chain constantregion (not including the stop codon) to generate a chimericimmunoglobulin gene. The resulting chimeric protein encodes an IgG1immunoglobulin molecule that is retained on the cell surface.

The H2kk transmembrane domain sequence can be modified via the insertionof flanking LoxP sites (as indicated below) to create a construct whichconverts a surface-expressed antibody into a secreted antibody upon theregulated expression of cre recombinase. In the nucleic acid sequencebelow (SEQ ID NO: 451), the C-terminal portion of constant domain of theIgG heavy chain is shown, indicating the locations of the 2 loxP sites(underlined) flanking the H2kk transmembrane domain (capital letters).Relevant restriction sites are boxed.

Corresponding amino acid sequence for the modified loxP modifiedtransmembrane domain is shown below. LoxP sites are shown underlined;sequence after the stop codon (*) is not shown.

(SEQ ID NO: 452) . . . LAPSSKSTSGGTAALGCLVKDYFPEPVTVSWNSGALTSGVHTFPAVLQSSGLYSLSSVVTVPSSSLGTQTYICNVNHKPSNTKVDKKVEPKSCDKTHTCPPGPAPELLGGPSVFLFPPKPKDTLMISRTPEVTCVVVDVSHEDPEVKFNWYVDGVEVHNAKTKPREEQYNSTYRVVSVLTVLHQDWLNGKEYKCKVSNKALPAPIEKTISKAKGQPREPQVYTLPPSRDELTKNQVSLTGLVKGFYPSDIAVEWESNGQPENNYKTTPPVLDSDGSFFLYSKLTVDKSRWQQGNVESCSVMHEALHNHYTQKSLSLSPGKLEITSYSIHYTKLSPPPSTVSNMA TVAVLVVLGA*

Expression of cre recombinase in the cell leads to the recombination andloss of the transmembrane domain resulting in the in situ creation of asecreted form of the protein in the transfected cell population whichcan then used for further studies.

Cre recombinase(Accession numbers: P06956, AAY72404, and YP_006472) (SEQID NO: 453) MSNLLTVHQNLPALPVDATSDEVRKNLMDMFRDRQAFSEHTWKMLLSVCRSWAAWCKLNNRKWFPAEPEDVRDYLLYLQARGLAVKTIQQHLGQLNMLHRRSGLPRPSDSNAVSLVMRRIRKENVDAGERAKQALAFERTDFDQVRSLMENSDRCQDIRNLAFLGIAYNTLLRIAEIARIRVKDISRTDGGRMLIHIGRTKTLVSTAGVEKALSLGVTKLVERWISVSGVADDPNNYLFCRVRKNGVAAPSATSQLSTRALEGIFEATHRLIYGAKDDSGQRYLAWSGHSARVGAARDMARAGVSIPEIMQAGGWTNVNIVMNYIRNLDSETGAMVRLLEDGD Accession number. NC_005856(from YP_006472). (SEQ ID NO: 454)atgtccaatttactgaccgtacaccaaaatttgcctgcattaccggtcgatgcaacgagtgatgaggttcgcaagaacctgatggacatgttcagggatcgccaggcgttttctgagcatacctggaaaatgcttctgtccgtttgccggtcgtgggcggcatggtgcaagttgaataaccggaaatggtttcccgcagaacctgaagatgttcgcgattatcttctatatcttcaggcgcgcggtctggcagtaaaaactatccagcaacatttgggccagctaaacatgcttcatcgtcggtccgggctgccacgaccaagtgacagcaatgctgtttcactggttatgcggcggatccgaaaagaaaacgttgatgccggtgaacgtgcaaaacaggctctagcgttcgaacgcactgatttcgaccaggttcgttcactggaaaatagcgatcgctgccaggatatacgtaatctggcatttctggggattgcttataacaccctgttacgtatagccgaaattgccaggatcagggttaaagatatctcacgtactgacggtgggagaatgttaatccatattggcagaacgaaacgctggttagcaccgcaggtgtagagaaggcacttagcctgggggtaactaaactggtcgagcgatggatttccgtctctggtgtagctgatgatccgaataactacctgttttgccgggtcagaaaaaatggtgttgccgcgccatctgccaccagccagctatcaactcgcgccctggaagggatttttgaagcaactcatcgattgatttacggcgctaaggatgactctggtcagagatacctggcctggtctggacacagtgcccgtgtcggagccgcgcgagatatggcccgcgctggagtttcaataccggagatcatgcaagctggtggctggaccaatgtaaatattgtcatgaactatatccgtaacctggatagtgaaacaggggcaatggtgcgcctgctggaagatggcgattag.

The corresponding nucleic acid sequences corresponding to these genes,set forth below, are made by DNA 2.0 (Menlo Park, Calif.), and correctsynthesis is confirmed by sequence analysis.

The nucleic acid clones are provided in DNA2.0 vectors (i.e. pJ31 orpJ51), which are devoid of most 6 bp restriction endonucleaserecognition sites. For the purposes of the construction of theimmunoglobulin library, suitable vectors must not include any of thefollowing restriction sites: AclI (AACGTT; SEQ ID NO: 350), AscI(GGCGCGCC; SEQ ID NO: 351), BbsI (GAAGAC [SEQ ID NO: 352]; GTCTTC [SEQID NO: 353]), BsrnBI (CGTCTC [SEQ ID NO: 354]; GAGACG [SEQ ID NO: 355]),EagI (CGGCCG; SEQ ID NO: 356), FseI (GGCCGGCC; SEQ ID NO: 357), MfeI(CAATTG; SEQ ID NO: 358), NheI (GCTAGC; SEQ ID NO: 359), SbfI (CCTGCAGG;SEQ ID NO: 360), SgrAI (CRCCGGYG; SEQ ID NO: 361).

The poly nucleotides sequences of the heavy chain variable domainscaffolds used for the construction of one embodiment of thepolynucleotide libraries of the invention are provided below. In thesesequences, the following landmarks for the heavy chain sequences are:

boxed sequence; Kozak, unboxed capital letters;

IGHV, small letters;

boxed capital letters; initial small stuffer for CDR3 (underlined smallletters);

boxed capital letters; IgG1 constant region, small letters; XhoI, boxedcapital letters; transmembrane domain and cytoplasmic tail, unboxedcapital letters; AscI cloning site, boxed capital letters.

Heavy Chain Variable Regions

Sequence H11; SEQ ID NO: 362. IgHV 1-2-stuffer region-IgG1 constant-H2kkperitransmembrane, transmembrane and cytoplasmic domains. (1570 bp)(Vh1). Landmark restriction sites are shown in boxed letters. (Thestuffer region, shown in underlined small letters, was replaced by bonafide CDR3 region sequences obtained by PCR of human peripheral bloodlymphocyte RNA.)

boxed letters; Kozak consensus sequence, unboxed capital letters;IGKV4-1 variable sequences, small letters;

restriction site, boxed capital letters; small initial stuffer for cdr3,underlined small letters;

restriction site, boxed capital letters; k constant region, smallletters;

sit restriction site, boxed capital letters.

Lambda Light Chains.

boxed small letters; Kozak consensus sequence, unboxed capital letters;IGLV1-40 variable region, small letters;

restriction site, boxed capital letters (note that this is a class IIrestriction site which cleaves outside of its recognition site, overhangof GGCT is indicated by underlined and bolded capital letters); smallinitial stuffer for cdr3, underlined small letters;

boxed capital letters; IGLC3 lambda constant region, small letters;

Bold-underline in L1 indicates unwanted BbsI that needs to be mutated

Cloning

The heavy chain, kappa light chain, and lambda light chain libraries areassembled in the vector pJ31 starting with the H1, L1 and K1 variabledomains, as shown in FIG. 21. Each prototypic library contains (a) areplaceable variable region (Vh1, Vκ1, or Vλ1); (b) a small stufferregion bounded by restriction sites into which synthetic, orPCR-amplified heavy chain, κ light chain and λ light chain derived CDR3regions are inserted; (See Examples 6, 7 and 8) and (c) the heavy chain,κ light chain and λ light chain constant regions respectively. (Thesecloning vectors and the overall cloning strategy are shown schematicallyin FIG. 21A, FIG. 21B, and FIG. 21C, respectively).

To minimize background ligation of cut vectors, a longer stuffer of 671bp is cloned into the EagI+NheI sites of plasmid pJ31-Vh1 to generatevector pJ31-Vh1s (FIG. 21A step 2), into the BsmBI+MfeI sites ofpJ31-Vκ1 to generate pJ31-Vκ1s (FIG. 21B step 7), and into the BbsI+AclIsites of pJ31-Vλ1 to generate pJ31-Vλ1s (FIG. 21C step 12).

This step facilitates the excision of double-cut vector away from anyvector that might be only singly or incompletely double-cut, from apreparative agarose gel to minimize background during the CDR3 cloningstep.

The sequences H2 (Vh2) through H9 (Vh9), containing heavy chain variableregions flanked by restriction sites SgrAI and EagI, are cloned in placeof H1 (Vh1) in vector pJ31-Vh1s to generate constructs pJ31-Vh2sthrough-Vh9s (FIG. 21A step 3); Similarly variable kappa light regionsK2-K5 (Vκ2-Vκ5) are cloned in place of K1 (Vκ1) to generate constructspJ31-Vκ2s through-Vκ5s (FIG. 21B step 8); and variable lambda lightchains L2-L4 (Vλ2-Vλ4) are cloned in place of L1 (Vλ1) to generateconstructs pJ31-Vλ2s through-Vλ4s (FIG. 21C step 13).

Thus this process creates a total of 18 intermediate cloning vectors, inwhich each of the 9 heavy chain scaffolds, 5 kappa light chain scaffoldsand 4 lambda light chain scaffolds were inserted into the plasmid pJ31.These intermediate plasmids were then used to introduce PCR amplifiedCDR3, and can also be used to introduce fully synthetic CDRs asdescribed below.

The theoretical diversity resulting from such a library construct isgreatly reduced relative to that expected using all possible variableregions. In present library, a potential 11,016 (9V×204D×6J) (IGHV)heavy chains may be observed after artificial recombination andassembly. Likewise, there is a potential for 25 (5V×5J) kappa chains and28 (4V×7J) lambda chains that might be observed. This leads, ignoringpotential non-templated mutations at the domain junctions, for a totalpredicted complexity of 583848 members (11016H×(25K+28L)). In contrast,67320 heavy chain (55V×204D×6J), 220 kappa chains (44V×5J), and 273lambda chains (39V×7J) would be expected for the complete representationof the human IgG naïve locus, for a total theoretical complexity of3.318×10⁷ members. Therefore, we have maintained the total antigenbinding capability of our library, while reducing its total complexityby an estimated 56.8 fold (3.318×10⁷/583848), providing for robust andredundant presentation of all library members for selection.

Example 6 PCR Amplification of CDRs Preparation of OligonucleotidePrimers Specific for the CDR3 Region

The choice of a primer's nucleotide sequence depends in general onfactors such as the distance on the nucleic acid from the region codingfor the desired sequence, its hybridization site on the nucleic acidrelative to any second primer to be used, the number of genes in therepertoire it is to hybridize to, and the like.

Primers are designed and selected within the 3′ end of the framework 3region and the 5′ end of the appropriate constant domains to enable theCDR3 regions of Ig λ, κ, and H chain isoforms to be PCR amplified.Restriction sites are chosen based on several criteria: The restrictionsites must be as close as possible, but not overlap, the CDR3 regions;but should not include any known sites in the germline sequences of theD and J regions that compose the CDR3, such that digestion of the PCRproduct would reduce the overall diversity of the library and should becompatible with the cloning vectors to which they are to be inserted,specifically for example, the vectors described in Examples 3, 4 and 5above; and as outlined in FIG. 21.

The diversity-enriched naturally occurring CDR3 regions are generatedvia PCR mediated amplification as described below:

Total RNA from seven different donors is isolated from the peripheralblood monocytes (PBMC's; AllCells, Inc., Emeryville, Calif.) and pooled.

HPLC purified oligonucleotides are ordered from Allele Biotech, (SanDiego, Calif.). Total RNA is reverse transcribed using oligos H8a, H9a,K7a, L8a (as described below in Table 14) to generate amplified cDNA toIgG, IgM, Igκ and Igλ, respectively, using the Superscript 3 protocol asprovided by Invitrogen Corporation (Carlsbad, Calif.).

Double-stranded cDNA copies of the highly diverse CDR3 populations areamplified by PCR using combinations of sense and antisenseoligonucleotides listed below using standard PCR amplificationconditions.

Heavy Chain Oligonucleotides for PCR Amplification

H1b. ggaatcCGGCCGtgtattactgtgcaaga (heavy chain sense oligo for IGHV6-1,EagI site; SEQ ID NO: 380) H2b. ggaatcCGGCCGtgtattactgtgcgaaa (heavychain sense oligo for IGHV3-30-3, EagI site; SEQ ID NO: 381) H3b.ggaatcCGGCCGtgtattactgtgcgaga (heavy chain sense oligo for IGHV4-34 (1mismatch), IGHV3-7) (1 mismatch), IGHV4-59, IGHV1-69, IGHV1-2, andIGHV5-51 (1 mismatch, , EagI site; SEQ ID NO: 382) H4b.ggaatcCGGCCGtatattactgtgcgaaa (heavy chain sense oligo for IGHV3-23,EagI site; SEQ ID NO: 383) H5b. ggaatcgGCTAGCgggaagacggatgggcccttg(heavy chain antisense from IgG constant, NheI site; SEQ ID NO: 384)H6b. ggaatcgGCTAGCgggaagaccgatgggcccttg (heavy chain antisense from IgGconstant, NheI site; SEQ ID NO: 385) H7b.ggaatcgGCTAGCgggaaaagggttggggcgga (heavy chain antisense from IgMconstant, NheI site; SEQ ID NO: 386) H8a. gaagtagtccctgaccaggc (reversetranscription primer for IgG; SEQ ID NO: 387) H9a. aagtcctgtgcgaggcagc(reverse transcription primer for IgM; SEQ ID NO: 388)

Restriction sites are shown in capital letters.

Kappa light chain oligonucleotides for PCR amplification

K1b. ggaatcCGTCTcgTATtactgtcagcaatattatag (kappa CDR3 sense for IGKV4-1,BsmBI site; SEQ ID NO: 389) K2b. ggaatcCGTCTCgTATtactgtcagcagtatggtag(kappa CDR3 sense for IGKV3-20, BsmBI site; SEQ ID NO: 390) K3b.ggaatcCGTGTCgTATtactgcatgcaaggtacaca (kappa CDR3 sense for IGKV2D-30,BsmBI site; SEQ ID NO: 391) K4b. ggaatccGTCTCgTATtactgtcaacagagttacag(kappa CDR3 sense for IGKV1D-39, BsmBI site; SEQ ID NO: 392) K5b.ggaatcCGTCTCgTATtactgtcaacagtatgataa (kappa CDR3 sense for IGKV1-33,BsmBI site; SEQ ID NO: 393) K6b. ggaatcCAATTGctcatcagatggcgggaag (kappaCDR3 antisense for all kappa light chains (MfeI site; SEQ ID NO: 394)K7a. ggcctctctgggatagaag (kappa CDR3 reverse transcription primer forIgK; SEQ ID NO: 395).

Lambda light chain oligonucleotides for PCR amplification

L1b. ggaatcGAAGACGAGGCTgattattactgccagtcct (lambda CDR3 sense forIGLV1-40 with BbsI site; SEQ ID NO: 396) L2b.ggaatcGAAGACGAGGCTgattattactgctgctcat (lambda CDR3 sense for IGLV2-11with BbsI site; SEQ ID NO: 397) L3b.ggaatcGAAGACGAGGCTgactattactgtcaggtgt (lambda CDR3 sense for IGLV3-21with BbsI site; SEQ ID NO: 398) L4b.ggaatcGAAGACGAGGCTgagtattactgcctgctct (lambda CDR3 sense for IGLV7-43with BbsI site; SEQ ID NO: 399) L5b. ggaatcAACGTTaccgtggggttggccttg(lambda CDR3 antisense from constant with AcII site; SEQ ID NO: 400)L6b. ggaatcAACGTTaccgagggggcagccttg (lambda CDR3 antisense from constantwith AcIIsite; SEQ ID NO: 401) L7b. ggaatcAACGlTaccgatggggcagccttg(lambda CDR3 antisense from constant with AcII site; SEQ ID NO: 402)L8a. gctcccgggtagaagtcac (reverse transcription primer for lambda CDR3(primes all lambda light chains); SEQ ID NO: 403)

PCR conditions are as follows:

Kappa, PCR condition A used for K2b, K3b, with K6b:

-   -   95° C.×3′ for 1 cycle; then 95° C.×30,″ 60° C.×30,″ 72° C.×30″        for 3 cycles; then 95° C.×30,″ 70° C.×30,″ 72° C.×30″ for 30        cycles; then 72° C.×5′.

Kappa, PCR condition B used for K1b, K4b and K5b with K6b:

-   -   95° C.×3′ for 1 cycle; then 95° C.×30,″ 55° C.×30,″ 72° C.×30″        for 3 cycles; then 95° C.×30,″ 68° C.×30,″ 72° C.×30″ for 30        cycles; then 72° C.×5′.

PCR Conditions for Lambda:

-   -   95° C.×3′ for 1 cycle; then 95° C.×30,″ 58° C.×30,″ 72° C.×30″        for 3 cycles; then 95° C.×30,″ 66° C.×30,″ 72° C.×30″ for 33        cycles; then 72° C.×5′.

PCR conditions for IgG and IgM:

-   -   95° C.×3′ for 1 cycle; then 95° C.×30,″ 60° C.×30,″ 72° C.×30″        for 3 cycles; then 95° C.×30,″ 68° C.×30,″ 72° C.×30″ for 33        cycles; then 72° C.×5′.

The final total diversity in PCR amplified products from each of thesereactions is directly related to the antibody repertoire in the humanimmune system, which is very great. Indeed, the bands resulting from PCRappear as a smear on agarose gels in the range of 100 to 200 bp. Theranking of thickness of the visualized bands (i.e. how heterogenous thepopulation sizes were) was mu>gamma>kappa=lambda.

The sequence of 34 independent IgM-derived CDR3 clones that resultedfrom PCR using oligos H2b+H7b was obtained (data not shown). No twosequences were identical, and the insert coding size ranged from 3 to 27amino acids.

After successful PCR amplification of the CDR3 regions, the resultingPCR products are gel purified and the restriction endonuclease digestedproducts are ligated into the antibody heavy chain, kappa light chainand lambda light chain scaffolds in the pJ31 cloning vectors asdescribed in Example 7 in place of the pre-existing stuffer fragments(FIG. 21A step 4, FIG. 21B step 9, and FIG. 21C step 14, respectively),as described below.

TABLE 14 Reverse Ig transcription species reaction # primer oligo 1oligo 2 amplified 1 H8A H5b H1b IgG 2 H8A H6b H1b IgG 3 H8A H5b H2b IgG4 H8A H6b H2b IgG 5 H8A H5b H3b IgG 6 H8A H6b H3b IgG 7 H8A H5b H4b IgG8 H8A H6b H4b IgG 9 H9A H7b H1b IgM 10 H9A H7b H2b IgM 11 H9A H7b H3bIgM 12 H9A H7b H4b IgM 13 K7a K6b K1b kappa 14 K7a K6b K2b kappa 15 K7aK6b K3b kappa 16 K7a K6b K4b kappa 17 K7a K6b K5b kappa 18 L8a L5b L1blambda 19 L8a L6b L1b lambda 20 L8a L7b L1b lambda 21 L8a L5b L2b lambda22 L8a L6b L2b lambda 23 L8a L7b L2b lambda 24 L8a L5b L3b lambda 25 L8aL6b L3b lambda 26 L8a L7b L3b lambda 27 L8a L5b L4b lambda 28 L8a L6bL4b lambda 29 L8a L7b L4b lambda

Example 7 Ligation of PCR Amplified CDRs into Antibody Scaffolds

A. Ligation and Sub Cloning of the PCR Amplified CDRS into the Heavy andLight Scaffolds is Accomplished as Described Below:

Transformation into bacteria is accomplished via electroporation usingthe protocol as follows: Ligated DNA (5-40 ng) is electroporated into 20μL of EP-Max 10b electrocompetent cells from BioRad (Hercules, Calif.)in a 0.1 cm gap cuvette using BioRad's Gene Pulser XCell Electroporatorwith settings of 1.8 kV, capacitance of 25 μF, and 200 ohms ofresistance. Following electroporation, 600 μL SOC was added to each tubeand entire contents were plated on a 15 cm ampicillin-containing agarplate.

The IgG and IgM PCR amplified CDR3s from Example 6 above, and theintermediate 9 heavy chain cloning vectors from Example 5 (i.e.VH₁₋₉-stuffer-IgG constant region) are digested with the restrictionenzymes EagI+NheI (FIG. 21A step 4) and gel purified.

The kappa light chain PCR amplified CDR3s from Example 6 above, and theand the 5 κ light chain intermediate cloning vectors from Example 5 arecut with the restriction enzymes BsmBI+MfeI (FIG. 21B step 9) and gelpurified.

The lambda light chain PCR amplified CDR3s from Example 6 above, and the4λ light chain intermediate cloning vectors from Example 5 are cut withthe restriction enzymes BbsI+AclI (FIG. 21C step 14) and gel purified.

All digested CDR3s are then ligated into their appropriate intermediatecut cloning vectors to generate completed heavy chain, kappa light andlambda light chains semi synthetic polynucleotide sub libraries (i.e.stuffer regions have been replaced with the highly diverse,PCR-amplified CDR3 regions).

Prior to ligation the intermediate cloning vectors from Example 5 can bepooled, for example the 9 heavy chain vectors (VH₁₋₉-stuffer-IgGconstant region), the 5 κ light chain vectors (Vκ₁₋₅-stuffer-IGKC kappaconstant region), and the 4λ light chain vectors (V_(λ1-4)-stuffer-IGLC3lambda constant region) can be pooled into 3 separate pools (i.e. onepool each of heavy-, κ-, and λ-intermediate cloning vectors) prior toaddition and ligation of CDR3's. Alternatively, the vectors can be keptseparate, in which case one can set up 18 separate ligations.

The 9 separate (or pool) of complete heavy chain polynucleotide libraryvectors containing the highly diverse naturally occurring CDR3collection described above, are digested and then sub-cloned into theSgrAI and FseI sites of the final eukaryotic episomal expression vector,pABLh (FIG. 21A step 5).

The complete kappa and lambda light chain polynucleotide libraries arekept as two independent pools. Each set of inserts, κ and λ, aredigested and then sub-cloned into the SbfI and AscI sites of the finaleukaryotic episomal expression vectors, pABLκ (FIG. 21B step 10) andpABLλ (FIG. 21C step 15).

The integrity and diversity of the library is confirmed by sequencingthe CDR3 inserts from a representative and statistically significantnumber of clones (i.e., 50 to 200 samples from each of the heavy, κ andλ chain sub libraries).

Plasmid stocks of the library expression vectors were prepared usingstandard procedures and stored frozen, until required.

B. Creation of Cell Surface Expression Libraries

One day prior to transfection, HEK-293 cells are seeded at a density ofthree million cells per T75 flask in 10 mL of DMEM medium containing 10%fetal bovine serum. A total of 50 flasks are prepared for transfectionand subsequently incubated at 37° C., in a tissue culture incubator with5% carbon dioxide overnight.

The next day, a mixture of 30 mL OptiMEM (Invitrogen Corporation,Carlsbad, Calif.), 1.2 mL of HD-Fugene (Roche Diagnostics Corp.,Indianapolis, Ind.), 90 μg of Ig heavy chain DNA (i.e., vector pABLh)and 90 μg of Ig light chain (i.e., vector pABLκ and/or vector pABLλ) DNAare mixed and incubated for 25-30 minutes at room temperature. A volumeof approximately 540 μl is added to each T75 flask containing theHEK-293 cells and the cells are incubated at 37° C., 5% carbon dioxide.

Three days post-transfection, the cells are transferred to T225 flaskscontaining 25 mL DMEM medium containing 10% fetal bovine serum.Blasticidin (15 μg/mL), and puromycin (1.5/g/mL) are added after cellattachment in order to select for successfully transfected cells. Thecells are incubated at 37° C., 5% carbon dioxide during a selectionprocess of two-four weeks. During this time, cells are monitored forgrowth, the medium is exchanged and the cells are expanded intoadditional T225 flasks as required.

After selection, the cells are screened to confirm high level surfaceexpression of antibodies as described below and then used to create acell bank. Cell banks are created from pooling the cells from onehundred T225 flasks.

Cells are harvested by trypsinization treatment and then pelleted bygentle centrifugation. The cell pellets are resuspended in cell freezingmedium at a concentration of 5.8×10⁷ cells/ml. One mL of cells isdispensed into each of ninety cryovials. The vials are incubatedovernight at −80° C. and then transferred to liquid nitrogen forlong-term storage.

C. Creation of a Dynamic Cell Surface Antibody Library

One day prior to transfection, HEK-293 cells comprising an inducible orconstitutive AID expression vector with a hygromycin selectable markergene (as described in Example 3) are seeded at a density of threemillion cells per T75 flask in 10 mL of DMEM medium containing 10% fetalbovine serum. A total of 50 flasks are prepared for transfection andsubsequently incubated overnight at 37° C. in a tissue culture incubatorwith 5% carbon dioxide.

The next day, a mixture of 30 mL OptiMem, 1.2 mL of HD-Fugene, 90 μg ofIg heavy chain DNA (i.e., vector pABLh) and 90 μg of Ig light chain(i.e., vector pABLκ and or vector pABLλ) DNA are mixed and incubated for25-30 minutes at room temperature. A volume of approximately 540 μl isadded to each T75 flask containing the HEK-293 cells and the cells areincubated at 37° C., 5% carbon dioxide.

Three days post-transfection, the cells are transferred to T225 flaskscontaining 25 mL DMEM medium containing 10% fetal bovine serum and 50micrograms per mL of G418 antibiotic. Hygromycin (350 g/mL), blasticidin(15 μg/mL), and puromycin (1.5 μg/mL) are added after cell attachment inorder to select for successfully transfected cells. The cells areincubated at 37° C., 5% carbon dioxide during a selection process oftwo-four weeks. During this time cells are monitored for growth, themedium is exchanged and the cells are expanded into additional T225flasks as required.

Cells stably expressing heavy and light chain (i.e., functionalantibodies) from the initial selections above are characterized toestablish copy number of expressed antibody on the cell surface by FACS.Briefly fluorescently tagged antibodies to the heavy and light chain areused to stain samples of transfected cells from the library usingcommercially available fluorescein Isothiocyanate (FITC) orR-Phycoerythrin (R-PE) conjugated goat anti-human-IgG (Sigma). Stainingis performed using the manufacture's suggested protocols, usually viaincubation of the test cells in the presence of labeled antibody for 30minutes on ice. Expression levels are quantified using Bang Beads (BangLaboratories Inc., Fishers, Ind.) with five different microbeadpopulations with defined human IgG-binding capacities. The geometricmean fluorescence intensity of each population is determined by flowcytometry and plotted against their individual IgG-binding capacity togenerate a linear regression curve. This curve can then be used toconvert the geometric mean fluorescence of each cell line into anaverage IgG expression level. Heavy and light chains designed using themethods described herein are further elucidated in Example 13.

D. Creation of Cell Banks

Cell banks are created from pooling the cells from one hundred T225flasks. Briefly, cells are harvested from plates by trypsinization andthen pelleted by gentle centrifugation. The cell pellets are resuspendedin cell freezing medium at a concentration of 5.8×10⁷ cells/ml. One mLof cells is dispensed into each of ninety cryovials. The vials areincubated overnight at −80° C. and transferred to liquid nitrogen forlong-term storage.

E. Conversion of Surface Displayed Libraries to Secreted Libraries

Polynucleotides encoding the IgG1 constant region, a DNA fragment of aportion of the juxtamembrane, and complete transmembrane and cytoplasmicdomain from the murine histocompatibility 2, κ region (H2kk, NCBIaccession number AK153419) are synthesized by (DNA 2.0 Menlo Park,Calif.). Silent mutations are introduced during synthesis near the 5′end of the IgG constant region to create a convenient NheI site. TwoXhoI restriction sites are introduced by site directed mutagenesis; thefirst is introduced between the two synthetic fragments, and a secondXhoI restriction site and an adjacent in-frame stop codon are introduceddistal to the cytoplasmic domain sequence, as shown below.

Sequence of IgG1 constant region, with contiguous H2kk transmembranedomain.

Features as shown in 5′ to 3′ order are: NheI site (boxed smallletters), IgG1 constant region coding sequence (small letters), XhoIsite (introduced between the constant and juxtamembrane region, insmall, boxed letters); murine H2kk sequence that contains a smalljuxtamembrane region and transmembrane and cytoplasmic domains (capitalletters, the native stop codon is underlined), 2^(nd) XhoI site sequence(boxed and in capitals), and adjacent in-frame stop codon (smallunderlined letters); additional cloning sites are shown in smallletters, the EcoRI site used in later cloning steps is boxed anditalicized.

This fragment is reclaimed with NheI and EcoRI and cloned into thecognate sites of the expression vector ANA327 (vector format 1, withblasticidin resistance) using standard cloning methodology. Digestion ofvector with XhoI is followed by self-religation to remove thetransmembrane, juxtamembrane and cytoplasmic domains (the capitalletters in the sequence above) and generate the vector ANA346 for theproduction of secreted production of proteins. In this case, the secondstop codon (tga shown in underlined in the sequence above) serves as thein-frame translation stop for the secreted form of the IgG1 heavy chain.Cotransfection of the expression vector above, along with appropriateexpression vectors for the desired kappa or lambda light chain, in HEK293 cells permitted subsequent secretion of the protein into the tissueculture media in reasonable yield. The resultant secreted proteins canbe produced and purified to determine binding or functionalcharacteristics using standard methodology and as further describedherein.

As shown in Examples 4, 5 and 6 above, this library format enables adiverse repertoire of high affinity antibodies to be readily selectedand affinity matured. The results from screening this library indicatethat relatively low repertoire libraries of less than <10⁶ members canbe successfully used to create high affinity antibodies when combinedwith on-going hypermutation of the antibodies displaying the preferredbinding and/or functional characteristics.

Example 8 Synthetic CDRs

A synthetic polynucleotide sequence of the present invention is shownschematically in FIG. 22. In this example, a synthetic CDR3 domain thatcontains two circularly permuted WRC motifs containing preferred SHMcodons with the preferred hot spots (AGCTAC; SEQ ID NO: 404) iscontained within 2 nonameric ideal cold spots (GTCGTCGTC; SEQ ID NO:405) to create a boundary of SHM resistant sequence. As shown below, thereading frame context of the hot spots (bold) may be manipulated(underlining) so that the hot spot motif can be introduced into each ofthe available three reading frames.

SEQ ID NO: 406 GTCGTCGTCAGCTACAGCTACGTCGTCGTC . . . first readingframe;; SEQ ID NO: 407 GTCGTCGTCCAGCTACAGCTAGTCGTCGTC . . . secondreading frame;; and SEQ ID NO: 408 GTCGTCGTCACAGCTACAGCTGTCGTCGTC . . .third reading frame;.

As shown in FIG. 22, this synthetic CDR sequence provides an opportunityto demonstrate the ability of synthetic preferred SHM motifs toselectively drive targeted diversity generation at the amino acid level,while minimizing mutations in SHM resistant sequences. The systematicplacement of the hot spot in each reading frame demonstrates the impactof the reading frame context on amino acid mutation generation. Theconstruct further provides for the ability to for the elimination ofnon-mutated vectors through the use of a restriction enzyme whichrecognizes the native sequence making the analysis of mutated sequencesmore efficient (because non mutated sequences are eliminated). Inaddition, the experiment can be conducted in the absence of selectivepressure to select for, or against any specific type of mutationalevent.

A. Synthesis and Cloning

The complete polynucleotide sequence of one of the three synthetic CDR3antibody constructs is shown below. In this sequence the synthetic SHMoptimized sequence is shown in capitals; hot spots are shown as boldcapital letters and cold spots are shown as italicized capital letters.Also shown in the sequence below in bold, lowercase letters is thelocation of the BbsI+AclI restriction digestion sites that are used inExamples 3, 4, 5, 6, and 7 to ligate the PCR amplified,naturally-occurring, CDR3 sequences into the antibody scaffolds createdpreviously:

SEQ ID NO:409 atgaaacacctgtggttcttcctcctcctggtggcagctcccagatgggtcctgtcccaggtgcagctacaacagtggggcgcaggactgttgaagccttcggagaccctgtccctcacctgcgctgtctatggtgggtccttcagtggttactactggagctggatccgccagcccccagggaaggggctggagtggattggggaaatcaatcatagtggaagcaccaactacaacccgtccctcaagagtcgagtcaccatatcagtagacacgtccaagaaccagttctccctgaagctgagctctgtgaccgccgcggacacggccgtgtattactgtgcgagaGT CGTCGTCAG CTACAGCTACGTCGTCGTCgctgaatacttccagcactggggccagggcaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcaccctcctccaagagcacctctgggggcacagcggccctgggctgcctggtcaaggactacttccccgaaccggtgacggtgtcgtggaactcaggcgccctgaccagcggcgtgcacaccttcccggctgtccttcagtcctcaggactctactccctcagcagcgtggtgaccgtgccctccagcagcttgggcacccagacctacatctgcaacgtgaatcacaagcccagcaacaccaaggtggacaagaaagttgagcccaaatcttgtgacaaaactcacacatgcccaccgtgcccagcacctgaactcctggggggaccgtcagtcttcctcttccccccaaaacccaaggacaccctcatgatctcccggacccctgaggtcacatgcgtggtggtggacgtgagccacgaagacctgaggtcaagttcaactggtacgtggacggcgtggaggtgcataatgccaagacaaagccgcgggaggagcagtacaacagcacgtaccgggtggtcagcgtcctcaccgtcctgcaccaggactggctgaatggcaaggagtacaagtgcaaggtctccaacaaagccctcccagcccccatcgagaaaaccatctccaaagccaaagggcagccccgagaaccacaggtgtacaccctgcccccatcccgggatgagctgaccaagaaccaggtcagcctgacctgcctggtcaaaggcttctatcccagcgacatcgccgtggagtgggagagcaatgggcagccggagaacaactacaagaccacgcctcccgtgctggactccgacggctccttcttcctatacagcaagctcaccgtggacaagagcaggtggcagcaggggaacgtcttctcatgctccgtgatgcatgaggctctgcacaaccactacacgcagaagagcctc tccctgtctccgggtaaa;.

The actual synthetic sequences of all of the synthetic CDR3 sequences,used for cloning and construction is shown below:

SEQ ID NO:410 acacggccgtgtattactgtgcgagaGTCGTCGTCAGCTACAGCTACGTCGTCGTCgctgaatacttccagcactggggccagggcaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcac;. SEQ ID NO:411acacggccgtgtattactgtgcgagaGTCGTCGTCCAGCTACAGCTAGTCGTCGTCgctgaatacttccagcactggggccagggcaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcac;. SEQ ID NO:412acacggccgtgtattactgtgcgagaGTCGTCGTCACAGCTACAGCTGTCGTCGTCgctgaatacttccagcactggggccagggcaccctggtcaccgtctcctcagcctccaccaagggcccatcggtcttcccgctagcac;.

The corresponding nucleic acid sequences corresponding to thesesequences, set forth above, can be made by DNA2.0 (Menlo Park, Calif.),and correct synthesis confirmed by sequence analysis.

These sequences can be inserted into the heavy chain scaffolds describedpreviously, using the same methodology and cloning steps as described inthe heavy chains scaffolds as described in Examples 5, 6 and 7, with thenaturally occurring PCR amplified CDR3s.

B. Analysis and Testing

The ideal CDR3 hot spot, in each permutation shown above, contains asingle SfcI restriction site that enables the removal of all sequenceswithin a library population that have not undergone SHM at thatposition. This simplifies and speeds up analysis by eliminating nonmutated sequences from being rescued and cloned.

To establish selective mutagenesis and diversity generation in theconstructs, the following steps are followed.

1. Transfection of Cells

Hek 293 cells are plated to a density of about 4×10⁵/well, in 6-wellmicrotiter dish. After 24 hours, transfections are performed usingFugene6 reagent from Roche Applied Sciences (Indianapolis, Ind.) at areagent-to-DNA ratio of 3 μL:1 μg DNA per well with the expressionvectors comprising the synthetic heavy chains and representative lightchain which confer blasticidin and hygromycin resistance respectively.Transfections are carried out in accordance with manufacturer'sprotocol.

Cells stabling expressing synthetic heavy chain constructs are createdusing standard methodology as described above, and are characterized toestablish copy number of expressed antibody on the cell surface by FACS.Briefly fluorescently tagged antibodies to the heavy and light chain areused to stain transfected cells and those exhibiting a copy number ofgreater than 500,000 intact heavy and light chains are selected.

Staining of light and heavy chain expression can be accomplished, forexample, by using commercially available fluorescein isothiocyanate(FITC) or R-Phycoerythrin (R-PE) conjugated rat anti-mouse Ig, kappalight chain, and FITC or R-PE conjugated rat anti-mouse Ig G1monoclonalantibodies (BD Pharmingen). Staining can be performed using themanufacturer's suggested protocols, usually via incubation of the testcells in the presence of labeled antibody for 30 minutes on ice.

Expression levels can be quantified using Spherotech rainbow calibrationparticles (Spherotech, Ill.) that enables the quantitative analysis ofcellular antigen expression to be determined.

Cells stably expressing heavy and light chain at a high level can beisolated by FACS sorting using standard flow and sorting protocols, andselected cells can be subsequently grown up for use as substrates foranalysis.

Selected cells expressing heavy and light chains as described above canthen be transfected with an expression vector containing an inducible,cold AID using standard transfection conditions as described above.Three days post transfection, selective pressure is exerted, and a newstable cell population is propagated that includes the inducible AIDexpression vector.

This population of cells is grown up, and AID expression is induced viathe addition of tetracycline or an analog thereof for about 6 to 24hours. The cells are allowed to expand for about 2 to 5 days, and thecycle repeated 2 to 5 times to generate diversity within the syntheticCDRs.

After an appropriate time, for example 2 to 3 weeks, vectors can berescued from the cells and the corresponding heavy chain cDNA sequencesanalyzed to determine the pattern of mutations achieved with eachsynthetic CDR. For example, a PCR amplified fragment can be digestedwith SfcI, and then the fragment is reamplified to permit the cloning ofDNA in which the SfcI site has been eliminated, presumably due to theaction of AID.

2. Episomal Rescue

As episomal vectors remain unintegrated and easily separable from a hostcell's chromosomal material, plasmids can be recovered by the method ofHirt (Hirt, 1967; Kapoor and Frappier, 2005; Yates et al., 1984),transformed into competent bacteria and further manipulated to verifythe sequence, identity and/or properties of the encoded polypeptides.

Using an estimate of an average of 3 resident episomes of 8000 basepairs (bp) each per cell, one can expect a yield of approximately 30picogram (pg) per million cells (see, e.g., Formula 1). Assuming atransformation efficiency into electrocompetent E. coli of 10⁷ coloniesper μg of relaxed circle DNA, one can expect approximately 300 E. colicolonies, each representing a single recovered episome, to result permillion mammalian cells.

(10⁶ cells×3 episomes/cell)×(660 g/mol/bp)×(8000 bp/episome)×(10⁶colonies/μg)×(10⁶ μg)÷(6×10²³ episomes/mol)=2.6×10⁻¹¹ g (DNA per 10⁶cells).  Formula 1

Plasmids can also be recovered using a standard alkaline lysisprocedure, e.g., as per a protocol from Qiagen, Inc. (for procedure, seee. g.,www1.qiagen.com/literature/handbooks/PDF/PlasmidDNAPurification/PLS_QP_Miniprep/1034641_HB3QIAprep_(—)112005.pdf; and Wade-Martins et al., Nuc Acids Res 27:1674-1682 (1999)). In oneaspect, transfected mammalian cells are treated the same way as the E.coli described in the Qiagen protocol. Episomes present in the finaleluate are transformed into competent E. coli as described above. Usingeither the Hirt supernatant or alkaline lysis method requires beginningwith a significant cell population for isolating resident episomes. Inone non-limiting example, starting with 50,000 clonally derived cells,one might expect to obtain 10 to 20 recovered episomes as manifested incolonies of transformed E. coli.

Another standard method to characterize transfected genes, whetherepisomal or integrated, involves performing a Polymerase Chain Reaction(PCR) reaction directly on the relevant cell population followed bycloning and characterizing individual resulting PCR fragments. Thismethod has the advantage of not requiring a large starting population ofcells. PCR amplification of the resident active antibody open readingframe can successfully be performed on as little as a single cell. Thishas the effect of foreshortening the time from isolation of a cell ofinterest to the point of sequencing the responsible open reading frame.

Still another option is to perform Reverse Transcriptase-PolymeraseChain Reaction (RT-PCR) on the isolated cells thus identifying andcharacterizing the resident polypeptide(s) via expressed mRNA.

Alternatively the library can be used as the starting point for theaffinity maturation of an antibody to any specific target antigen orepitope of interest, for example as described in Example 9 below.

Example 9 Selection and Affinity Maturation of an Antibody from aNucleic Acid Library

As described previously, antibodies provide a natural template throughwhich somatic hypermutation can be applied to create mutant proteinswith enhanced properties. Such improved antibodies can be selected basedupon affinity selection, for example via FACS or via binding to magneticbeads.

A. Synthesis and Cloning.

The construction of prototypic heavy and light chain and light chainconstructs is described in Examples 4-8.

In order to express antibody on the cell surface, the heavy chain iscreated as a chimeric molecule with a murine H2kk (MHC type I)peri-transmembrane domain, transmembrane domain, and cytoplasmic domain.The H2kk sequences are determined from accession number AK153419 at theNational Center for Biotechnology Information (NCBI) nucleotidedatabase.

The nucleotide sequence of the full length chimeric, cell-surfaceassociated scaffold heavy chain is listed in Example 5. The nucleotidesequences of the scaffold kappa and lambda heavy chains are shown inExample 5.

1. Transfection of Cells

Hek 293 cells are plated at a density of 4×10⁵/well, in 6-wellmicrotiter dish. After 24 hours, transfections are performed usingFugene6 reagent from Roche Applied Sciences (Indianapolis, Ind.) at areagent-to-DNA ratio of 3 μL:1 μg DNA per well with the desiredcombinations of heavy chain and light chain expression vectors. Heavyand light chain expression vectors confer blasticidin and hygromycinresistance respectively. Transfections are carried out in accordancewith manufacturer's protocol.

2. Selection by Peptides

A simple test case, antibodies can be selected against the wellcharacterized antigen hen egg lysozyme (HEL). An unlabeled andbiotinylated monomeric peptide sequence that comprises the majority ofthe hen egg lysozyme (HEL) binding surface is synthesized. Dimericpeptide sequences can also be synthesized to compare whether presentingthe peptide as a dimer would enhance antibody binding by increasing theavidity of the antibody-peptide interaction. A tandem dimer and abranched multiple antigenic peptide (MAP) dimer can also tested.Peptides as well as biotinylated or unlabeled HEL protein can be coupledto paramagnetic polystyrene microparticle surfaces that had beenmodified with functional groups or coated with streptavidin (Invitrogen,1600 Faraday Ave., PO Box 6482, Carlsbad, Calif. 92008).

3. Coupling HEL Protein and Peptides to Tosylactivated Microparticles

The HEL protein and peptides are coupled to 2.8 micron Tosylactivatedparamagnetic polystyrene microparticles in a 1.5 ml microcentrifuge tube(Nilsson κ and Mosbach K. Eur. J. Biochem. 1980:112: 397402). Themicroparticles (2e09 microparticles/milliliter) are washed andresuspended in 100 mM borate buffer, pH 9.5 at a concentration of 1e09microparticles/ml. Eleven nanomoles of peptide or 6 ug/ml HEL are addedto the microparticles and the microparticle/peptide mixture wasincubated at room temperature for at least 48 hours with slow tiltrotation. After incubation, the supernatant is removed and themicroparticles are washed with 1 ml phosphate buffered saline solution(PBS), pH 7.2 containing 1% (weight/volume) BSA. Finally, themicroparticles are resuspended in 1 ml PBS solution, pH 7.2 containing1% (weight/volume) BSA.

4. Coupling Biotinylated HEL Protein and Peptides toStreptavidin-Conjugated Microparticles

Another option is to couple biotinylated peptides to paramagneticpolystyrene microparticles whose surfaces have been covalently linkedwith a monolayer of streptavidin. Briefly, the streptavidinmicroparticles are washed, resuspended in 1 ml PBS solution, pH 7.2containing 1% (weight/volume) BSA and 33 picomoles of biotinylatedpeptide or approximately 10 ug/ml biotinylated HEL are added to themicroparticle solution. The microparticle/peptide solution can beincubated for 30 minutes at room temperature with slow tilt rotation.After coupling, the microparticles can be washed and resuspended to afinal microparticle concentration of 1e09 microparticles/ml. (Argaranaet al. 1986; 14(4):1871-82; Pahler et al. J Biol Chem 1987:262(29):13933-7).

5. Cell Selection

Transfected HEK 293 cells are screened in order to isolate cells thatbind to the peptide-conjugated paramagnetic microparticles. A similarcontrol cell line that does not express antibody is used as a negativecontrol for the selections.

The cells are washed with an equal volume of PBS solution, pH 7.2 andresuspended in PBS solution, pH 7.2 containing 1% (weight/volume) BSA toa final cell concentration of 1e07 cells/ml. The cells are pre-clearedby adding 1e06 naked microparticles to the cells and incubating on arotator at 4° C. for 30 minutes. The unbound cells are gentlytransferred to a new tube. Peptide-conjugated or naked microparticles(1e07) are transferred into the tube with the cells and thecell:microparticle mixture can be incubated on a rotator at 4° C. for 30minutes. The unbound cells are removed and the mnicroparticle: cellmixture can be washed with cold PBS/1% BSA. The microparticles andattached cells are resuspended in 100 ul cell culture medium and can begrown initially in one well of a 96-well plate. The number ofmicroparticle-bound cells can be determined and the cells expanded untilthe next round of selection. The number of microparticle-bound cellsselected on the peptide-conjugated microparticles is compared with cellsbound to the naked microparticles and to the cells that do not expressantibody.

6. In Vitro Affinity Maturation

Cells stabling expressing heavy and light chain (i.e. are expressingfunctional antibodies) from the initial selections above arecharacterized to establish copy number of expressed antibody on the cellsurface by FACS. Briefly fluorescently tagged antibodies to the heavyand light chain are used to stain transfected cells and those exhibitinga copy number of greater than 500,000 intact heavy and light chains areselected.

Staining of light and heavy chain expression can be accomplished, forexample, by using commercially available fluorescein Isothiocyanate(FITC) or R-Phycoerythrin (R-PE) conjugated rat anti-mouse Ig, kappalight chain, and FITC or R-PE conjugated rat anti-mouse Ig G1 monoclonalantibodies (BD Pharmingen). Staining can be performed using themanufacture's suggested protocols, usually via incubation of the testcells in the presence of labeled antibody for 30 minutes on ice.

Expression levels can be quantified using Spherotech rainbow calibrationparticles (Spherotech, Ill.) that enables the quantitative analysis ofcellular antigen expression to be determined.

Cells stably expressing heavy and light chain at a high level can beisolated by FACS sorting using standard flow and sorting protocols, andselected cells can be subsequently grown up for use as substrates foraffinity maturation.

Selected cells expressing heavy and light chains as described above canthen be transfected with an expression vector containing an inducible,cold AID polynucleotide sequence using standard transfection conditionsas described above. Three days post transfection, selective pressure isexerted, and a new stable cell population is propagated that includesthe inducible AID expression vector.

This population of cells is grown up, and AID expression is induced viathe addition of tetracycline or an analog thereof for about 6 to 24hours. The cells are allowed to expand for about 2 to 5 days, and thenselected using the HEL protein or peptide coupled beads as describedabove.

Cells that preferentially and/or selectively bind to the HEL protein orpeptides with a higher affinity are selected and allowed to expand. Ifrequired, another round of AID induction and mutagenesis is repeated, asdescribed above, and again cells that exhibit improved, selective, andhigh affinity binding, are retained for further propagation and growth.

The new improved variants obtained can be further characterized asdescribed herein, and the sequence of the heavy and light chainsdetermined after RT-PCR, or episome rescue, as described in Example 8.

Example 10 Application of SHM Libraries to the Directed Evolution ofEnzyme Pathways

The evolution of bacteria with resistance to existing therapeuticregimens has sparked interest in the discovery and development of novelantibiotics. Ideal candidates for further research are those that actvia multiple modes of action, making resistance significantly moredifficult to attain. One such antibiotic is Nisin.

Nisin is a natural product of Lactococcus lactic, a lantibotic with abroad spectrum of activity against Gram-positive bacteria, commonly usedin food preservation against such pathogens as Listeria monocytogenesand Clostridium botulinum. (Bavin et al., Lancet. 1952 Jan. 19; 1(3):127-9)) Nisin is a ribosomally translated and post-translated peptide,which despite decades of use by the food industry, has not seen theinduction of common resistance mechanisms. This finding is likely aresult of two facts: one, the mode of action of Nisin biocidal activitycomes from its binding to Lipid II and secondary induction of poreformation, (Breukink et al., (2006)). Lipid II is a bacterial cell-wallcomponent that is not easily modified by Gram-positive bacteria andwhose use forms a rate-limiting step in the generation of the bacterialcell wall. Nisin also acts to inhibit spore formation.

Nisin is currently in preclinical development for the treatment ofseveral bacterial pathogens. It displays a spectrum of activity towardsseveral pathogens, including multi drug-resistant Streptococcuspneumoniae, vancomycin-resistant Enterococcus faecium, and Strepococcuspyogenes, all areas where new therapeutics are desperately needed(Goldstein et al., (1998)). In one study, Nisin was shown to be 8-16times more potent in the treatment of S. pneumoniae (in mice) thanvancomycin (Brumfitt et al., 2002).

Despite these promising features, Nisin and other lantibotics sufferfrom several important limitations. Bacteria, even closely related(isogenic) species, display a significant variation in their sensitivityto Nisin and other lantibiotics. Secondly, Nisin is cleared quickly frommammalian circulatory system. For Nisin to become a truly efficacioustherapeutic, it will need to have improved pharmacodynamic propertieswith a broad spectrum of biocidal activity. Here we discuss applicationof SHM to engineer a Nisin with improved qualities.

Biosynthesis of bioactive Nisin has been to shown to be dependent ononly five L. lactis proteins, NisA, NisB, NisC, NisP, and NisT (Kuiperset al., 2004, Rink et al., (2005)). NisA encodes for a precursor peptidewhich is dehydrated at several serine and threonine positions by NisB,leading to a modified peptide that is cyclized at five positions byNisC. Finally the pro-antibiotic has its leader peptide cleaved byprotease NisP, and is excreted to the media by transporter NisT (SeeFIG. 23) The five thioester rings, each catalyzed by NisC, are termedlanthionines, and define the lantibiotic family of modified peptideantibiotics.

The modular nature of this pathway, easy assay for bioactivity, broadspecificity and activity of the dehydratase and cyclase NisB and NisC,make this an ideal target for SHM driven co-evolution to produce novelantibiotic constructs. In one approach such a strategy could be based onmaking certain genes, or portions of genes more susceptible to SHM,while making other genes, or portions of those genes, resistant to SHM.

The amino acid sequences of the 5 genes involved in Nisin biosynthesisare shown below: In these sequences, bold residues indicate thosepositions to be made hot to SHM, while underlined residues are those tobe made cold to SHM.

NisA, Native Gene>NisA|gi|530218|gb|AAA26948.1|nisin[Lactococcuslactis]; SEQ ID NO: 413MSTKDFNLDLVSVSKKDSGASPRITSISLCTPGCKTGALMGCNMKTATCH CSIHVSK NisC, NativeGene>NisC|gi|44045|emb|CAA48383.1| nisC[Lactococcus lactis]; SEQ ID NO:414 MRIMMNKKNIKRNVEKIIAQWDERTRKNKENFDFGELT LST GLPGIILMLAELKNKDNSKIYQKKIDNYIEYIVSKLSTYGLLTGS LYS GAAGIALSILHLREDDEKYKNLLDSLNRYIEYFVREKIEGFNLENITPPDYD VIE GLSGILSYLLLINDEQYDDLKILIINFLSNLTKENNGLISLYIKSEN QM SQSESEM YPLGCLNM GLAHGLAGVGCILAYAHIKGYSNEASLSALQKIIFIYEKFEL ERKKQFLW KDG LVADELKKEKVIREASFIRDAWCYG GPGISLLYLYGGLA LDNDYFVDKAEKILESAMQRKL GIDSYMICHGYSGLIEICSLFKRLLNTK KFDSYMEEFNVNSEQILEEYGDESGTGFLEGISGCILVLSKFEYSINFTYWRQALLLFDDFLKGGKR NisB, Native Gene>gi|473018|emb|CAA79468.1|NisBprotein[Lactococcus lactis]; SEQ ID NO: 415MIKSSFKAQPFLVRNTILSPNDKRSFTEYTQVIETVSKNKVFLEQLLLANPKLYNVMQKYNAGLLKKKRVKKLFESIYKYYKRSYLRSTPFGLFSETSIGVFSKSSQYKLMGKTTKGIRLDTQWLIRLVHKMEVDFSKKLSFTRNNANYKFGDRVFQVYTINSSELEEVNIKYTNVYQIISEFCENDYQKYEDICETVTLCYGDEYRELSEQYLGSLIVNHYLISNLQKDLLSDFSWDTFLTKVEAIDEDKKYIIPLKKVQKFIQEYSEIEIGEGIEKLKEIYQEMSQILENDNYIQIDLISDSEINFDVKQKQQLEHLAEFLGNTTKSVRRTYLDDYKDKFIEKYGVDQEVQITELFDSTFGIGAPYNYNHPRNDFYESEPSTLYYSEEEREKYLSMYVEAVKNHNVINLDDLESHYQKMDLEKKSELQGLELFLNLAKEYEKDIFILGDIVGNNNLGGASGRFSALSPELTSYHRTIVDSVERENENKEITSCEIVFLPENIRHANVMHTSIMRRKVLPFFTSTSHNEVQLTNIYIGIDEKEKFYARDISTQEVLKFYITSMYNKTLFSNELRFLYEISLDDKFGNLPWELIYRDFDYIPRLVFDEIVISPAKWKIWGRDVNNKMTIRELIQSKEIPKEFYIVNGDNKVYLSQENPLDMEILESAIKKSSKRKDFIELQEYFEDENIINKGQKGRVADVVVPFIRTRALGNEGRAFIREKRVSVERREKLPFNEWLYLKLYISINRQNEFLLSYLPDIQKIVANLGGKLFFLRYTDPKPHIRLRIKCSDLFLAYGSILEILKRSQKNRIMSTFDISIYDQEVERYGGFDTLELSEAIFGADSKIIPNLLTLIKDTNNDWKVDDVSILVNYLYLKCFFQNDNKKILNFLNLVSPKKVKENVNEKIEHYLKLLKVDNLGDQIFYDKNFKELKHAIKNLFLKMIAQDFELQKVYSIIDSIIHVHNNRLIGIERDKEKLIYYTLQRLFVSEEYMK NisP, NativeGene>gi|730155|sp|Q07596|NISP_LACLA Nisin leader peptide-processingserine protease nisP precursor; SEQ ID NO: 416MKKILGFLFIVCSLGLSATVHGETTNSQQLLSNNINTELINHNSNAILSSTEGSTTDSINLGAQSPAVKSTTRTELDVTGAAKTLLQTSAVQKEMKVSLQETQVSSEFSKRDSVTNKEAVPVSKDELLEQSEVVVSTSSIQKNKILDNKKKRANFVTSSPLIKEKPSNSKDASGVIDNSASPLSYRKAKEVVSLRQPLKNQKVEAQPLLISNSSEKKASVYTNSHDFWDYQWDMKYVTNNGESYALYQPSKKISVGIIDSGIMEEHPDLSNSLGNYFKNLVPKGGFDNEEPDETGNPSDIVDKMGHGTEVAGQITANGNILGVAPGITVNIYRVFGENLSKSEWVARAIRRAADDGNKVINISAGQYLMISGSYDDGTNDYQEYLNYKSAINYATAKGSIVVAALGNDSLNIQDNQTMINFLKRFRSIKVPGKVVDAPSVFEDVIAVGGIDGYGNISDFSNIGADAIYAPAGTTANFKKYGQDKFVSQGYYLKDWLFTTANTGWYQYVYGNSFATPKVSGALALVVDKYGIKNPNQLKRFLLMNSPEVNGNRVLNIVDLLNGKNKAFSLDTDKGQDDAINHKSMENLKESRDTMKQEQDKEIQRNTNNNFSIKNDFHNISKEVISVDYNINQKMANNRNSRGAVSVRSQEILPVTGDGEDFLPALGIVCISILGILKRKTKN NisT, NativeGene>gi|44044|emb|CAA48382.1|nisT [Lactococcus lactis]; SEQ ID NO: 417MDEVKEFTSKQFFYTLLTLPSTLKLIFQLEKRYAIYLIVLNAITAFVPLASLFIYQDLINSVLGSGRHLINIIIIYFIVQVITTVLGQLESYVSGKFDMRLSYSINMRLMRTTSSLELSDYEQADMYNIIEKVTQDSTYKPFQLFNAIIVELSSFISLLSSLFFIGTWNIGVAILLLIVPVLSLVLFLRVGQLEFLIQWQRASSERETWYIVYLLTHDFSFKEIKLNNISNYFIHKFGKLKKGFINQDLAIARKKTYFNIFLDFILNLINILTIFAMILSVRAGKLLIGNLVSLIQAISKINTYSQTMIQNIYIIYNTSLFMEQLFEFLKRESVVHKKIEDTEICNQHIGTVKVINLSYVYPNSNAFALKNINLSFEKGELTAIVGKNGSGKSTLVKIISGLYQPTMGIIQYDKMRSSLMPEEFYQKNISVLFQDFVKYELTIRENIGLSDLSSQWEDEKIIKVLDNLGLDFLKTNNQYVLDTQLGNWFQEGHQLSGGQWQKIALARTFFKKASIYILDEPSAALDPVAEKEIFDYFVALSENNISIFISHSLNAARKANKIVVMKDGQVEDVGSHDVLLRRCQYYQELYYSEQYEDNDE

NisB, NisP and NisT

As described above, the creation of SHM resistant “cold” versions of theessential genes NisP and NisT means that these genes will tend to mutateat a lower rate than SHM susceptible genes that are targeted fordiversity generation. Both NisP and NisT currently have broadspecificity for the Nisin and do not add to the potential diversity ofthe post-translationally modified peptide. In this initial example, NisBis also made SHM resistant; however it could also be selectively mutatedfollowing the same guidelines outlined below for NisA. Correspondingwild type and cold versions of these genes are shown in FIGS. 24, 25,26, 27 and 28.

NisA Peptide

As shown above, the majority of the leader peptide region of the NisApeptide should be made cold to SHM mediated mutagenesis because thissequence is absolutely necessary for substrate recognition by NisBCPT.The bulk of the remainder of the NisA peptide sequence should be madehot to SHM mediated mutagenesis, or alternatively, as shown above keyresidues involved in the generation of the lanthionines may be made SHMresistant thereby reducing the rate of their mutagenesis.

Corresponding wild type and cold versions of the NisA polynucleotidesequence are shown in FIG. 29. Codon optimization of NisA results in thecreation of 20 cold spots and elimination of all but one hot spot in theleader sequence, and the creation of 17 hot spots, compared to 8 hotspots in the wild type sequence, in the rest of the molecule.

NisC Protein

Regions of NisC involved in substrate recognition and cyclization, suchas those outlined above (bold residues, above and FIG. 23B), can be madehot to SHM mediated mutation, so that they have a greater probability ofgenerating mutants with alternate activities and specificities therebycreating mature Nisin molecules with altered modifications andbioactivity. Structural areas that govern only stability of the proteincan be made cold. Corresponding wild type and cold versions of the NisCpolynucleotide sequence are shown in FIGS. 30 and 31.

A specific example of the creation of a targeted hot spot in this geneis shown below.

In this example, an additional hot spot has been inserted into theregion of interest (LSTG) and a cold spot has been removed. Additionallythe flanking sequence has been made significantly more SHM resistant.

SEQ ID NO: 418 ..N..F..D..F..G..E..L..T.. L..S..T..G ..L..P..G aminoacid sequence; Native polynucleotide sequence:HhhhhhhhhhhhhhhhHhhhhhhhhhhhhHhhhhhhhhhhhhHhh hot spotscccccccCcccccCCcCccccCcCcCcCccccccCCccccccccc cold spots Optimizedpolynucleotide sequence: HhhhhhhhhhhhhhhhHhhhhhhhhhhHhHhhhhhhhhhhhhhhhot spots ccccccCcccccCCcCccCcccCcccCcccccccCcCcCCccCc cold spots

After final review to ensure that the synthetic polynucleotide sequenceis free of extraneous restriction sites, the complete syntheticpolynucleotide sequences can be synthesized (DNA 2.0, Menlo Park,Calif.), and cloned appropriate cloning vectors and sequenced to confirmcorrect synthesis.

Synthetic genes may then be introduced into expression vectors andtransformed into an appropriate bacterial strain, for example aLactococcus lactis strains as previously described (Mota-Meira et al.,1997) together with AID, (Besmer et al., 2006) or an AID homolog such asan Apobec-1 enzyme.

Screening may be accomplished by allowing the SHM mediated generateddiversity to evolve L. lactis co-cultured with Gram-positive bacterialtargets that are currently poorly targeted by Nisin. Eventually strainsof L. lactis will evolve that comprise mutated Nisin genes with enhancedactivity against the chosen bacterial target.

Mass spectroscopy of the supernatant of evolved cell-cultures can beused to assess the progress of the process (i.e. identified novellantibiotics with improved activity to a pathogen).

Example 10 References

-   1. Brumfitt W, Salton M R, Hamilton-Miller J M. Nisin, alone and    combined with peptidoglycan-modulating antibiotics: activity against    methicillin-resistant Staphylococcus aureus and vancomycin-resistant    enterococci.-   2. J Antimicrob Chemother. 2002 November; 50(5):731-4.-   3. BAVIN E M, BEACH A S, FALCONER R, FRIEDMANN R. Nisin in    experimental tuberculosis Lancet. 1952 Jan. 19; 1(3):127-9.-   4. Mota-Meira M, Lacroix C, LaPointe G, Lavoie M C. Purification and    structure of mutacin B-Ny266: a new lantibiotic produced by    Streptococcus mutans. FEBS Lett. 1997 Jun. 30; 410(2-3):275-9.-   5. Goldstein B P, Wei J, Greenberg K, Novick R. Activity of nisin    against Streptococcus pneumoniae, in vitro, and in a mouse infection    model. J Antimicrob Chemother. 1998 August; 42(2):277-8.-   6. Breukink E, de Kruijff B. Lipid II as a target for antibiotics.    Nat Rev Drug Discov. 2006 April; 5(4):321-32.-   7. Li. B, Yu J P, Brunzelle J S, Moll G N, van der Donk W A, Nair    S K. Structure and mechanism of the lantibiotic cyclase involved in    nisin biosynthesis. Science. 2006 Mar. 10; 311(5766):1464-7.-   8. Besmer E, Market E, Papavasiliou F N. The transcription    elongation complex directs activation-induced cytidine    deaminase-mediated DNA deamination. Mol Cell Biol. 2006 June;    26(11):4378-85.-   9. Kuipers A, de Boef E, Rink R, Fekken S, Kluskens L D, Driessen A    J, Leenhouts K, Kuipers O P, Moll G N. NisT, the transporter of the    lantibiotic nisin, can transport fully modified, dehydrated, and    unmodified prenisin and fusions of the leader peptide with    non-lantibiotic peptides. J Biol. Chem. 2004 May 21;    279(21):22176-82.-   10. Rink R, Kuipers A, de Boef E, Leenhouts κ J, Driessen A J, Moll    G N, Kuipers O P. Lantibiotic structures as guidelines for the    design of peptides that can be modified by lantibiotic enzymes.    Biochemistry. 2005 Jun. 21; 44(24):8873-82.

Example 11 The Design of Synthetic Libraries for Rapid Evolution ofEnzymes Via Somatic Hypermutation Zinc-Finger Proteins ExhibitingAltered DNA-Binding Specificity

Transcription factors bind to DNA and RNA and are located in the nucleusof eukaryotic cells. Transcription factors are candidates for somatichypermutation as described herein to optimize the activity of thefactors.

There are several families of transcription factors in eukaryoticorganisms, of which, Cys₂His₂ zinc finger proteins are the most common.Zinc finger domains are stabilized by a single zinc metal coordinated bytwo histidine and two cysteine residues. Each domain containsapproximately 30 amino acid residues; and each domain contains a mixedβ-sheet-α-helix secondary structure, with residues in the α-helixmediating DNA or RNA binding contacts (FIG. 32). Proteins are commonlyorganized in tandem arrays of fingers, with each finger binding anadjacent tri-nucleotide sub-site (FIG. 32) or region within the majorDNA or RNA groove, and with specific amino acids making specific DNA orRNA base contacts (see, FIG. 33, for example).

Transcription factors with engineered DNA-binding specificity provide apowerful and broadly applicable technology with scientific andtherapeutic functions. For example, zinc finger proteins exhibitingspecificity for a gene target could enhance or inhibit transcription, orsequester an mRNA message yet to be translated. Likewise, fusion of azinc-finger protein with a protein domain containing, for example, anenzymatic, therapeutic, or diagnostic activity could provide anotherproductive avenue for design of diagnostic and therapeutic proteins.Examples of zinc finger proteins include, but are not limited to, thosethat bind and fluoresce in recognition of a cancer specific DNA lesionor target a therapeutic moiety to a specific genomic region. In oneaspect, zinc finger nucleases (ZFNs), have the potential to be apowerful tool for targeting genome alteration in plants, insects, andhumans. ZFNs combine an engineered zinc finger protein joined to anon-specific endonuclease domain, capable of introducing double-strandedlesions that stimulate homologous and non-homologous recombination. Theability to modify a specific genomic region or target therapeutics ofinterest has utility in vitro and ex vivo research and gene therapyapplications. The application of this technology relies on the abilityto design zinc finger domains targeted to a genomic locus of interest.

The structures of numerous native and designed zinc-finger DNA complexeshave been determined by x-ray crystallography and rudimentary rules havebeen established that describe the recognition of a DNA trinucleotidemotif by a single zinc finger (Wolfe S A, Grant R A, Elrod-Erickson M,Pabo C O Beyond the “recognition code”: structures of two Cys2His2 zincfinger/TATA box complexes Structure (2001) 9(8):717-23.). Rational, insilico design of zinc finger proteins that bind larger DNA motifscontinues to be studied and various library approaches have beenemployed to create and select for larger binding sites with higherspecificity, some involving multiple rounds of selection and constructmanipulation (Rebar et al. 1994). In order to target binding of a zincfinger protein exhibiting specificity to a single region of the Homosapiens genome, a recognition site of at least about 18 nucleotides istypically required. As each finger can utilize up to 4 or 5 amino acidsto bind a tri-nucleotide motif and a static library of up to (20⁴)⁵, or˜11×10²⁶ members, is needed to find an optimal DNA-binding sequence,well beyond the complexity of phage or ribosomal libraries (Roberts, RW, 1999).

Application of SHM to libraries of zinc finger proteins capable ofundergoing targeted mutagenesis and selection provides an ideal solutionto this design problem. Because somatic hypermutation can generate novelmutations at desirable and undesirable locations (one or more codons)not initially present in a library, several strategies are available forthe generation and selection of novel binding proteins.

Each finger of a protein is composed of regions that are essential fortheir structural stability. To the extent possible, residues in thesepositions should be made cold to SHM to avoid mutations that couldresult in loss of function. An example of this is illustrated in FIG.33: positions that must be conserved for zinc finger binding function tobe retained are the cysteine and histidine residues that bind the zincmetal, and conserved aromatic and hydrophobic amino acids. In thisexample, the amino acid Valine precedes each cysteine in the fingershown in FIG. 33. Neither the valine or cysteine plays any role in DNAbinding and recognition. Eight possible codon combinations can be usedto encode these two amino acids. Scoring all possible 4096 bi-codoncombinations, the hexanucleotide combination GTGTGC ranks 4060^(th) of4096 possible sequences in its ability to recruit SHM; i.e., a “coldspot” to SHM and can be preferentially selected for preventing SHMmediated mutagenesis.

Each zinc finger contains regions and residues that are involved inbinding to DNA via direct amino acid, nucleotide base contacts and theseare, typically, the positions that are varied in static libraries tocreate binding variants. Two possible strategies are available forgenerating diversity at these positions using SHM, in combination orseparately with static library approaches, as discussed below:

In a first approach of zinc finger design, it is feasible to identify aclose variant of an existing zinc finger DNA-binding construct such asthat seen in FIG. 32. In one aspect, an existing binding zinc finger isto be varied in order to bind a DNA sequence that differs at only oneDNA base or at a single binding sub site. In this instance, one couldcreate and select zinc finger binding variants that differ at only oneor a few amino acid positions within a single finger, or within alocalized region. In one non-limiting embodiment, using an existing zincfinger sequence optimized for recruitment of SHM-mediated mutagenesis,while making the remaining, invariant fingers cold to SHM, representsone design. For example, FIG. 32 shows a closer view of finger 1, wheresuccessive residues glutamate (E) and histidine (H) make contacts to theDNA. Four possible hex peptides encode EH, one of which, GAGCAC (SEQ IDNO: 419), is significantly more “hot” (susceptible) to SHM-mediatedmutagenesis than the other three possibilities. Silent substitutions tothe underlying DNA code that create “hot spots” for SHM are desirable.Similarly, silent “cold spot” substitutions to the DNA-binding residuesand regions of zinc fingers 2 and 3 which are expected to remaininvariant during the course of selection can also be employed.

A second approach to library design is the introduction of “preferredhot spot SHM codons” at sites known to mediate DNA-binding contacts andat which diversity should be generated. One finding of the analysis ofSHM “hot spots” is that some SHM hot spot motifs presented in thereading frame of reference plays a role in the generation of diversity.As shown in FIG. 1 and FIG. 2 the same SHM-mediated mutagenesis activityspectrum acting on the same hot spot motifs (under selective pressure),produces different outcomes when viewed withincomplementarity-determining regions (FIG. 1) and framework regions (FIG.2) of immunoglobulin heavy and light chains. The basis for this findingis that the codon reading frame of reference for the hot spot has animpact on whether an induced mutation is silent (a change in codon thatproduces no change in amino acid, most common in framework regions) orwhether the mutation produces amino acid diversity. As a consequence ofthis observation, certain codons, such as AGC (serine), TAT, (tyrosine),TAC (tyrosine), and AAC (asparagine), when arranged in randomlyassembled libraries (FIG. 3 (WAC) and FIG. 5 (WRC)), generate tightlyinterleaved hot spots that are natural generators of amino aciddiversity, as seen in affinity matured antibodies (FIG. 4 and FIG. 6). Asimilar approach may then be applied to library design of zinc-fingerarrays. The regions known to contribute to DNA binding and specificity,particularly the n-terminal residues of each fingers alpha helix, may beconstructed entirely from these simplified codon alphabets. As can beseen in FIG. 4 and FIG. 6, this approach, when paired with SHM-mediatedmutagenesis rapid generates a diversity of amino acids (15 of the 20amino acids) at each position. If we contrast this approach with themore typical construction of static libraries on a three zinc-fingerconstruct, the differences in the resulting library complexity areclear. A simple NNK codon-based library, with 5 NNK library positionsper finger and a total of three fingers, would have: (4*4*2)

(5*3)=3.77*10²² potential members. In contrast, a WRC libraryrepresentation of the same zinc finger library, with 5 randomizedpositions over three fingers, will have only 2

(5*3)=32768 members. The difference, then, is a static library thatcannot be even partially represented using any selection techniques,versus SHM-based libraries that can easily and redundantly be presentedusing a standard selection methods. Finally, these WAC and WRC librarymethodologies may be paired with strategies, as outlined above, formaking functionally conserved and important regions cold to SHM-mediatedmutagenesis.

Example 11-References

-   1. Bae, K. H., Do Kwon, Y., Shin, H. C., Hwang, M. S., Ryu, E. H.,    Park, K. S., Yang, H. Y., Lee, D. K., Lee, Y., Park, J., Sun Kwon,    H., Kim, H. W., Yeh, B. I., Lee, H. W., Hyung Sohn, S., Yoon, J.,    Seol, W. & Kim, J. S. (2003) Human zinc fingers as building blocks    in the construction of artificial transcription factors Nat.    Biotech. 21, 275-80.-   2. Bae, K. H. & Kim, J. S. (2006) One-step selection of artificial    transcription factors using an in vivo screening system Mol Cells    21: 376-380.-   3. Jamieson, A. C., Miller, J. C. & Pabo, C. O. (2003) Drug    Discovery with Engineered Zinc-Finger Proteins Nature Reviews Drug    Discovery 2, 361-368.-   4. Hurt, J. A., Thibodeau, S. A., Hirsh, A. S., Pabo, C. O. &    Joung, J. K. (2003) Highly specific zinc finger proteins obtained by    directed domain shuffling and cell-based selection Proc Natl Acad    Sci USA 100, 12271-6.-   5. Greisman, H. A. & Pabo, C. O. (1997) A general strategy for    selecting high-affinity zinc finger proteins for diverse DNA target    sites Science 275, 657-61.-   6. Joung, J. K., Ramm, E. I. & Pabo, C. O. (2000) A bacterial    two-hybrid selection system for studying protein-DNA and    protein-protein interactions Proc Natl Acad Sci USA 97, 7382-7.-   7. Rebar, E. J. & Pabo, C. O. (1994) Zinc finger phage: affinity    selection of fingers with new DNA-binding specificities Science 263,    671-3.-   8. Wolfe, S. A., Greisman, H. A., Ramm, E. I. & Pabo, C. O. (1999)    Analysis of zinc fingers optimized via phage display: evaluating the    utility of a recognition code J Mol Biol 285, 1917-34.-   9. Bibikova, M., Beumer, K., Trautman, J. K. & Carroll, D. (2003)    Enhancing gene targeting with designed zinc finger nucleases Science    300, 764.-   10. Bibikova, M., Golic, M., Golic, K. G. & Carroll, D. (2002)    Targeted chromosomal cleavage and mutagenesis in Drosophila using    zinc-finger nucleases Genetics 161, 1169-75.-   11. Porteus, M. H. & Baltimore, D. (2003) Chimeric nucleases    stimulate gene targeting in human cells Science 300, 763.-   12. Roberts, R. W. (1999) Totally in vitro protein selection using    mRNA-protein fusions and ribosome display Curr Opin Chem Biol    3(3):268-73.

Example 12

Design of Optimized Seed Libraries for SHM

Affinity matured antibodies were analyzed in order to characterizenucleotide motifs that recruit somatic hypermutation (SHM) to the siteof mutation, and to develop a set of predictive algorithms thatdetermine how any DNA codon, motif, or family of sequences may evolveover time. Application of these findings to in vitro SHM proteinevolution, construct and library design are discussed.

Materials and Methods

Identification of SEM Events

Human IGHV, IGKV, and IGLV germline antibody sequences and their allelicforms were assembled from multiple online sources, including the NCBI(www.ncbi.nlm.nih.gov/entrez/), the IMGT antibody database(imgt.cines.fr/), and the VBASE database of human antibody genes(vbase.mrc-cpe.cam.ac.uk/). A total of 232 IGHV, 56 IGKV, and 66 IGLVvariable domain germline alleles were identified. Additional structuralinformation, such as those codons falling within framework andcomplementarity-determining regions (CDRs), Kabat numbering, and thecanonical loop turn structures of CDRs were also annotated.

The sequences of human affinity matured antibodies were collected fromthe antibody database at the National Center for Bioinformatics (NCBI)on Apr. 1, 2007 which can be found at the following world wide web site:ftp.ncbi.nih.gov/blast/db/fasta/igSeqNt.gz.

Our strategy was to first identify the likely originating germlinesequence for each affinity matured antibody, followed by an analysis ofthose residues that undergone modification as a result of SHM-mediatedaffinity maturation. An un-gapped BLAST alignment between a potentialgermline antecedent and an affinity matured antibody was accepted if itprovided greater than 94% sequence identify over the entire length ofthe antibody variable region, provided a best match relative to otherpotential originating germline sequences, and the sequences were notidentical. Because this database contains a variety of antibodysequences (IgA, IgE, IgG, IgD, IgM and subtypes thereof.) from bothgermline and affinity matured antibodies, care was taken to identifyaccurately the likely changes that arose from SHM-mediated alterationsof germline IGHV, IGKV and IGLV sequences. Mutations identified at the5′ and 3′ portions (3 residues) of the coding region alignment were notconsidered further in this analysis.

In this manner, a total of 106909 IGHV, 24378 IGKV and 24965 IGLVmutations were identified in 12956, 4165 and 3811 alignments to germlinesequences, respectively.

Identifying DNA Hot Spots/Cold Spots for SHM

DNA sequences that promote or discourage SHM were identified in thefollowing manner: no assumptions were made regarding the size of the SHMhot and cold motif. Likewise, the position of a mutation relative to thesite of the motif was allowed to vary. For each mutation, identified asdescribed above, we selected a nucleotide ‘window’ around the site,usually 9 or 15 nucleotides in length, likely to encompass any motifresponsible for recruiting SHM machinery (activation-induced cytidinedeaminase (AID) and error-prone polymerases). Within each X-mernucleotide window, we searched exhaustively for all motifs of length k,where an occurrence includes those sequences that vary at up to cpositions within the k-mer motif.

Our measure for the statistical significance of SHM motif occurrencescompares the number of times a k-mer motif is observed (N_(s)) in all NX-mer mutation windows with how often it would be expected to occur atrandom (Np_(s)) (where N is the total number of mutations and pa is theprobability of observing one or more motif occurrences within each X-merwindow). A Markov chain was used to estimate p, for each k-mer motif asdescribed previously (Tompa 1999), using nucleotide transitionprobabilities taken from human germline IGHV sequences, shown below.

${{ij} = {\left\lfloor \begin{matrix}0.169 & 0.270 & 0.381 & 0.179 \\0.289 & 0.287 & 0.101 & 0.321 \\0.239 & 0.219 & 0.314 & 0.227 \\0.155 & 0.278 & 0.413 & 0.154\end{matrix} \right\rfloor \mspace{14mu} {where}\mspace{14mu} i}},{j \in \left\{ {A,C,T,G} \right\}}$

The difference in the number of observed to expected motifs occurrencesis given by N_(s)−Np_(s), where √{square root over (Np_(s)(1−p_(s)))}represents the standard deviation of Np_(s), and the z-score for eachmotif is given by

M _(s)=(N _(s) −Np _(s))/√{square root over (Np _(s)(1−p _(s)))}

where M_(s) is the number of standard deviations by which the observednumber of motif occurrences exceeds the expected value. This metric wasused to rank order all possible motifs that might recruit or repel SHM.

Results

Analysis of mutations originating from SHM in antibodies undergoingaffinity maturation led to several important insights. Preferrednucleotide sequences are used at hot spots to attract the SHM machinery(see for example, Tables 2, 3, 6 and 9), and these hot spots arepositioned specifically with regard to the codon reading frame. As shownin FIG. 34, the 3-mer nucleotide motif AGC represents a preferred sitefor somatic hypermutation events (i.e., one preferred SHM codon). InFIG. 34, the number of mutations observed in the analysis is shown asthe line graph in each sub-graph at each position of the codon in theAGC motif found in framework (FR), and complementarity determiningregions (CDR) for the heavy and light chains of antibodies. The fontsize for each nucleotide position of the motif shows how often eachnucleotide which serves as the first position of the codon readingframe. Within framework regions, no one reading frame dominates, whereaswithin CDRs, the first position (A) of the AGC SHM motif is almostuniversally used as the first position of the codon.

The result is that certain hot spot codons (and therefore amino acids)placed within a specific reading frame context account for the majorityof somatic hypermutation events and the resulting diversity created fromthese events. FIG. 35 shows the 20 most hot spot codon hypermutationtransition events within the FR and CDR regions of heavy chainantibodies, where the numbers labeling the arrows indicate how often acodon transition event was observed. The codons AGC and AGT (Serine),and to a lesser extent TAC and TAT (Tyrosine), account for ˜50% of theoriginating mutations observed in affinity matured antibodies. Use ofthese hot spot codons within the correct reading frame, combined withaffinity maturation leads to many fewer observed silent mutations withinCDRs (highlighted by dotted circles in FIG. 35). Also, secondary andtertiary SHM events starting from the AGC or TAC codons lead to thepotential creation of many of the 20 possible amino acids.

We developed a probabilistic Markov chain model for predicting thetemporal diversity generated by SHM which results from SHM acting on asingle nucleotide codon, degenerate codon or SHM motif. A Markov chainis a discrete-time stochastic process that can used to calculate allfuture time states of a system. At each point in time, the system mayhave changed states from the state the system was in the moment before,or the system may have stayed in the same state.

Formally, this can be written as:

Pr(X _(n+1) =χ|X _(η)=χ_(η) , . . . , X ₁=χ₁)=Pr(X _(n+1) =χ|X_(n)=χ_(n)).

Where X₁, X₂, X₃, . . . represent a sequence of random variables withthe Markov property, namely that, given the present state, the futureand past states are independent. The probability of going from one statei to state j in n time steps is defined as:

P ^((n)) _(ij) =Pr(X _(n) =|X ₀ =i)

And the single-step transition as

P _(ij) =Pr(X ₁ =j|X ₀ =I)

The possible values of X_(i) form a countable set S called the statespace of the chain. Markov chains are often described by a directedgraph, where the edges are labeled by the probabilities of going fromone state to the other states.

Changes of state are called transitions. In this example, we chose toapply this method to codons undergoing SHM, where the system may existin any one of 64 possible codon states, and where any codon state may beaccessible from a different codon if there is a non-zero probability ofa SHM event connecting those two states. Other equivalent methods,including Markov chain Monte Carlo (MCMC), continuous-time Markovchains, and hidden Markov models (HMM), may also be used to solve thistime-dependent evolution problem.

The system begins with a probability distribution of starting codonstates, whose total probability is equal to 1. For example, a systemstarting with AAA as the only starting state would be written in matrixform as: [AAA, AAC, AAG, TTG, TTT]=[1, 0, 0, . . . , 0, 0].

Likewise, a system starting with a degenerate codon composed of half AAAand half TTT would be written in matrix form as: [AAA, AAC, AAG, . . . ,TTG, TTT]=[0.5, 0, 0, . . . , 0, 0.5].

FIG. 36 shows such a transition matrix for the system, where the y-axisrepresents the starting codon state, and complete listing of allpossible codons and the x-axis represents the ending codon state given around of SHM-mediated evolution.

The matrix describing the systems transition probabilities between codonstates shown in FIG. 36 was derived from an analysis of SHM events inheavy and light chain antibody sequences (see Materials and Methods),where each column of the matrix has a normalized probability equal toone. Off-diagonal values represent possible transitions between codonstates, diagonal terms represent self-transitions (or non-transitions),and the total matrix has an off-diagonal normalized evolution rate of0.5% per generation (selected arbitrarily based on ideal literaturevalues). Transition frequencies are encoded by through the use of a greyscale; the actual numerical values are presented in FIGS. 36B, 36C, 36Dand 36E.

The marginal distribution Pr (Xn=x) is the distribution over states attime n, and the initial distribution is Pr (X₀=x). The evolution of theprocess through one time step is described by a standard by theequation:

Pr(X _(n) =j)=Σ_(rεs) p _(rj) Pr(X _(n−1) =r)=Σ_(rεs) p _(rj) ^((n))Pr(X ₀ =r).

where ‘n’ is an integer value and the starting codon distribution hasevolved over ‘n’ iterative rounds, cording to the given state transitionprobabilities.

This system therefore depicts how a SHM system, starting with a specificsequence would evolve over multiple rounds of evolution given anystarting codon probability distribution.

FIGS. 37-44 show the accumulation of codon states and theircorresponding amino acid frequencies as a function of various rounds ofSHM-mediated evolution given different sets of starting codonfrequencies.

FIGS. 37 and 38 show the evolution of the codon AGC (serine), apreferred SHM codon, and the resulting amino acid frequencies over 50rounds of SHM-mediated mutagenesis, as calculated in our Markov chainmodel. Within a few rounds of mutation, many other amino acids becomecommon (frequency scale as given for FIG. 36). This finding supportsFIG. 35, which shows that single codons and their amino acids, inparticular AGC/AGT (Serine) and TAC/TAT (Tyrosine), can be utilized bySHM to drive creation of most of the other amino acids in a naturalcontext.

By comparison, equivalent calculations starting from a TCG,non-preferred codon, also coding for serine, are shown in FIGS. 39 and40, and demonstrate that such non-preferred codons are not just poorersubstrates for SHM, but that they generate less diversity as a functionof time then do preferred SHM codons.

FIGS. 41 and 42 show the rapid evolution of a mixed AGC/TAC, “WRC motif”comprising preferred SHM codons for Serine and Tyrosine) that prescribesrapid and effective generation of amino acid diversity.

FIGS. 43 and 44 show the evolution of a GGT codon (glycine), with theimmediate evolution of codons arising from single mutation events, suchas GAT (aspartate), GCT (alanine), and AGT (serine). Secondary mutationevents acting on these new codons give rise to a tertiary set of codons.For instance, both AGT and GGT under SHM produce the codon AAT, leadingto acquisition of asparagine at this position.

These results confirm that by developing a complete understanding of theprobability that a codon will be subject to SHM, in conjunction withspecific insight into how these sequences are utilized to generate aminoacid diversity, enables the development of specific algorithms thatprovide for the predictive creation of diversity in a heterologoussystem undergoing SHM. As shown below, by combining this understandingwith knowledge of the most favorable positions for mutations actuallyidentified from a highly selected evolving system, it is possible todevelop a rapid and effective system for mutagenesis.

Example 13 HyHEL10 Example of SHM-Mediated Affinity Maturation

An advantage of this SHM-mediated approach to creating diversity is thatrelatively simple libraries can be used to create an exceptionally largerepertoire of sequences during selection and evolution. In order todemonstrate this approach, we affinity matured an existing antibody thathas been well described in the literature. HyHEL10 is a mouse antibodyfirst derived from a hybridoma to the antigen hen egg white lysozyme(HEL). The antigen-antibody complex has been fully characterizedthermodynamically and by an atom resolution crystal structure.

For example, the constructs listed in Table 15 define a set ofantibodies, and sequence variants thereof that have fully definedsequences and affinities, e.g., Pons et al., (1999) Protein Science8:958-68; and Smith-Gill et al., (1984) J. Immunology 132:963.

TABLE 15 Hen Egg Lysozyme antibody constructs Mutations DNA Sequence Kdkoff kon Light chain variants wt LC/wt HC GGC30-AAC31-AAC32-CTA333.93E−11 8.6E−05 2.2E+06 LC G30(silent)N31A/wt HCGGA30-GCT31-AAC32-CTA33 1.48E−09 8.29E−03 5.61E+06 N31G LC/wt HCGGC30-GGT31-AAC32-CTA33 2.78E−09 1.21E−02 4.33E+06 N31S LC/wt HCGGC30-AGC31-AAC32-CTA33 7.10E−10 9.70E−04 1.40E+06 N32S LC/wt HCGGC30-AAC31-AGC32-CTA33 1.00E−10 1.90E−04 1.90E+06 N32G LC/wt HCGGC30-AAC31-GGT32-CTA33 6.29E−10 2.85E−03 4.53E+06 N31SN32S/wt HCGGC30-AGC31-AGC32-CTA33 2.50E−09 6.10E−03 2.40E+06 LC L33(silent)/wt HCGGC30-AAC31-AAC32-TTA33 5.96E−11 9.33E−05 1.56E+06 N31D LC/wt HCGGC30-GAT31-AAC32-CTA33 1.1E−10 Heavy chain variants wt LC/Y50A HCGGG49-GCC50-GTA51 Not detectable wt LC/Y33A HC GAT32-GCC33-TGG34 2.0E−08 4.45E−02 2.13E+06 Mixed heavy and light chain variants LCN31G/Y33A HC see above  7.0E−06 LC N32G/Y33A HC see above 2.00E−08

Nucleotides in bold represent codons in which defined mutations weremade to introduce codons that have been optimized for SHM to enableefficient somatic hypermutation, compared to the “wild type” (HyHEL10)sequence (“wt”), as defined below. LC=Light Chain; HC=heavy Chain.

These positions are previously known to be important for binding, and tohave been naturally mutated from the corresponding germline sequenceduring somatic hypermutation. Specifically, the light chain sequence ofHyHEL10 contains the residue Asn31 located within CDR1 that makes athermodynamically important contact to the HEL antigen residue Lys96.The Gly31 mutant (codon GGT) of HyHEL10 has a dissociation constant ofaround 2.5 nM, whereas the Asp31 (codon GAT) mutant of HyHEL10 hasdissociation constant of around 110 pM, and the wild-type Asn31 (codonof HyHEL10 has a dissociation constant of around 30 pM. We subjected aclonal population of HyHEL10 Gly31 (GGT) mutants, presented on thesurface of HEK293 cells, to iterative rounds of FACS based selectionagainst 50 pM FITC-HEL in the presence of SHM as described below.

A. Synthesis and Cloning of (“wild type”) HyHEL10 Heavy and Light ChainConstructs

The prototypic HyHEL10 heavy chain and light chain expression vectorswere created by starting with an episomal expression vector, asdescribed in Example 4 (vector format 1; U.S. Application No.60/902,414, entitled “Somatic Hypermutation Systems”), and usingstandard molecular genetic manipulations as follows: the original coldpuromycin resistance marker in vector AB102 was replaced with cold bsdor with pur using the NgoMIV and XbaI restriction sites, to generate thevectors AB187 and AB197, respectively.

A slightly longer, transcriptionally more robust version of the CMVpromoter was exchanged for the original sequence found in AB102 usingNheI (the mcs2 restriction site most proximal to the CMV promoter) andSbfI (the most CMV-proximal mcs1 site). The original AB102 CMV promoterincluded 553 bp of the unmodified CMV sequence upstream from the first Tof the TATA box, while the AB187 and AB197 versions included 645 bpupstream from the first T of the TATA box.

The nucleotide sequences for the “wild type” HyHEL10 heavy and lightchains (Pons et al., (1999) Protein Science 8:958-68) (sequences below)were synthesized (DNA 2.0, (Menlo Park, Calif.)). For cloning purposes,the heavy chain was bordered by BglII and AscI restriction sites, andthe light chain was bounded by SacI and AscI restriction sites.

In order to express HyHEL10 IgG and its mutants thereof on the cellsurface, the heavy chain was created as a chimeric molecule with thefollowing features: Kozak consensus sequence; HyHEL10 heavy chainvariable region; full-length murine IgG1 constant region; XhoI site;Murine H2kk (MHC type I) peri-transmembrane domain, transmembrane domainand cytoplasmic domain. The H2kk sequences were determined fromaccession number AK153419 at the National Center for BiotechnologyInformation (NCBI) nucleotide database.

The nucleotide sequence of the full length chimeric, cell-surfaceassociated HyHEL10 heavy chain is as listed below:

In this sequence, the BglII site is underlined; Kozak sequence isunderlined and italicized; stop codon is underlined and bolded; XhoIsite is indicated by boxed nucleotides; double underlined sequences arederived from H2kk. The AscI cloning site 3′ to the TGA stop codon isindicated by italicized nucleotides.

The amino acid sequence of the chimeric, cell-surface associated HyHEL10heavy chain is as listed below. The two amino acids (Leu-Glu) encoded bythe synthetic XhoI site are marked by bold-and-underlined; thebold-underline Glu also represents the most amino proximal amino acid ofthe H2kk domain; double underline indicates the putative transmembranedomain; and the asterisk indicates a stop codon.

(SEQ ID NO: 421) MNKLLCCALVFLDISIKWTTQDVQLQESGPSLVKPSQTLSLTCSVTGDSITSDYWSWIRKFPGNRLEYMGYVSYSGSTYYNPSLKSRISITRDTSKNQYYLDLNSVTTEDTATYYCANWDGDYWGQGTLVTVSAAKTTPPSVYPLAPGSAAQTNSMVTLGCLVKGYFPEPVTVTWNSGSLSSGVHTFPAVLQSDLYTLSSSVTVPSSPRPSETVTCNVAHPASSTKVDKKIVPRDCGCKPCICTVPEVSSVFIFPPKPKDVLTITLTPKVTCVVVDISKDDPEVQFSWFVDDVEVHTAQTQPREEQFNSTFRSVSELPIMHQDWLNGKEFKCRVNSAAFPAPIEKTISKTKGRPKAPQVYTIPPPKEQMAKDKVSLTCMITDFFPEDITVEWQWNGQPAENYKNTQPIMNTNGSYFVYSKLNVQKSNWEAGNTFTCSVLHEGLHNHHTEK SLSHSPGK LEPPPSTVSNMATVAVLVVLGAAIVTGAVVAFVMKMRRRNTGGKGGDYALAPGSQTSDLSLPDCKVMVHDPHSLA*

The amino acid and nucleotide sequence of the (“wild type”) HyHEL10kappa light chain is provided below.

Amino acid sequence of the HyHEL10 kappa light chain. Asterisk indicatesstop codon.

(SEQ ID NO: 422) MNKLLCCALVFLDISIKWTTQDIVLTQSPATLSVTPGNSVSLSCRASQSIGNNLHWYQQKSHESPRLLIKYASQSISGIPSRFSGSGSGTDFTLSINSVETEDFGMYFCQQSNSWPYTFGGGTKLEIKRADAAPTVSIFPPSSEQLTSGGASVVCFLNNFYPKDINVKWKIDGSERQNGVLNSWTDQDSKDSTYSMSSTLTLTKDEYERHNSYTCEATHKTSTSPIVKSFNRNEC*

The nucleotide sequence of the HyHEL10 kappa light chain. Start and stopcodons are underlined. SacI and AscI cloning sites are bolded.

(SEQ ID NO: 423) GAGCTCACCACAATGAACAAGTTGCTGTGCTGCGCGCTCGTGTTTCTGGACATCTCCATTAAGTGGACCACCCAGGATATTGTGCTAACTCAGTCTCCAGCCACCCTGTCTGTGACTCCAGGAAATAGCGTCAGTCTTTCCTGCAGGGCCAGCCAAAGTATTGGCAACAACCTACACTGGTATCAACAAAAATCACATGAGTCTCCAAGGCTTCTCATCAAGTATGCTTCCCAGTCCATCTCTGGGATCCCCTCCAGGTTCAGTGGCAGTGGATCAGGGACAGATTTCACTCTCAGTATCAACAGTGTGGAGACTGAAGATTTTGGAATGTATTTCTGTCAACAGAGTAACAGCTGGCCTTACACGTTCGGAGGGGGGACCAAGCTGGAAATAAAACGGGCTGATGCTGCACCAACTGTATCCATCTTCCCACCATCCAGTGAGCAGTTAACATCTGGAGGTGCCTCAGTCGTGTGCTTCTTGAACAACTTCTACCCCAAAGACATCAATGTCAAGTGGAAGATTGATGGCAGTGAACGACAAAATGGCGTCCTGAACAGTTGGACTGATCAGGACAGCAAAGACAGCACCTACAGCATGAGCAGCACCCTCACGTTGACCAAGGACGAGTATGAACGACATAACAGCTATACCTGTGAGGCCACTCACAAGACATCAACTTCACCCATTGTCAAGAGCT TCAACAGGAATGAGTGTTGAGGCGCGCC

Mutants of these “wild type” heavy and light chains, as well as thegermline sequence, as described above, Table 15, were created using sitedirected mutagenesis using the QuickChange® Multi Site DirectedMutagenesis kit (Stratagene, Calif.); sequences were confirmed bysequencing.

B. Transfection of Cells

A stable HEK-293 cell line expressing the [N31G HC/wt HC] anti-HELimmunoglobulin and AID activity was generated by seeding a T75 cultureflask with 3×10⁶ HEK-293 cells in 10 mL DMEM medium containing 10% FBS(Invitrogen Corporation, Carlsbad, Calif.). The following day, 500 μLOptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 20 μL HD-Fugene(Roche Diagnostics Corporation, Indianapolis, Ind.), 1 μg of theoptimized AID expression vector, (Example 4) and 1.5 μg each of theheavy and light chain expression vectors were mixed and incubated forapproximately 25-30 minutes at room temperature. After incubation thismixture was added drop-wise to the cell culture medium.

Approximately three days post-transfection, the cell growth medium wasexchanged with 10 mL DMEM medium containing 10% FBS, 50 μg/mL Geneticin,10 μL/mL Antibiotic-Antimycotic Solution, 1.5 μg/mL puromycin, 15/g/mLblasticidin, and 350 μg/mL hygromycin (Invitrogen Corporation, Carlsbad,Calif.) and the cells were incubated for approximately four weeks withperiodic reseeding and exchange of the cell culture medium. At the endof the selection period, the cell culture was expanded, archived and aT75 cell culture flask was seeded with 3×10⁶ HEK-293 cells that wereexpressing the [N31G LC/wt HC] anti-HEL immunoglobulin and AID activityin 10 mL DMEM medium containing 10% FBS (Invitrogen Corporation,Carlsbad, Calif.). The following day, 500 μL OptiMEM (InvitrogenCorporation, Carlsbad, Calif.), 20 μL HD-Fugene (Roche DiagnosticsCorporation, Indianapolis, Ind.), and 3 μg of the AID expression vectorDNA described above, were mixed and incubated for approximately 25-30minutes at room temperature. After incubation, this mixture was addeddrop-wise to the cell culture medium. After approximately one week ofincubation, the original stable HEK-293 cell line expressing the [N31GLC/wt HC] anti-HEL immunoglobulin and AID as well as the culture thathas been transiently transfected with additional AID expression vectorwere prepared for cell sorting.

C. Selection of Higher Affinity Mutants:

The selected HEK-293 cell line expressing the [N31G LC/wt HC] anti-HELimmunoglobulin and AID activity as well as the culture that had beentransiently transfected with additional AID expression vector wereprepared for cell sorting by collecting the cells, washing with an equalvolume of PBS solution, pH 7.2 and resuspending 1e07 cells from eachculture in ice-cold PBS solution, pH 7.2 containing 1% (weight/volume)BSA and either 50 pM or 500 pM HEL-FITC at a final cell concentration of2e05 cells/mL.

Round 1

Hen Egg lysozyme (Sigma Aldrich, Mo.) was labeled with fluoresceiniosthiocyanate (FITC) using the EZ-Label™ FITC protein labeling kit(Pierce, Rockford, Ill.) following the manufacturers directions.

Following incubation for 30 minutes at 4° C., the cells were pelleted bycentrifugation and the volume reduced to 200 μL. After transfer tosterile 3 mL tubes, a 1:500 dilution of PE-conjugated goat-anti-mouseimmunoglobulin was added to the cells and incubation continued at 4° C.for 30 minutes. The cells were then pelleted by centrifugation andresuspended in 1 mL of sterile ice-cold PBS solution, pH 7.2 containing1% (weight/volume) BSA plus 2 nanograms/milliliter DAPI. LiveIgG-positive cells that were positive for FITC (excitation with a 150 mW488 nm laser, collection through a 528/38 filter) were isolated byfluorescence activated cell sorting (FACS) using a Cytopiea Influx CellSorter at a flow rate of approximately 10,000 events/second (FIG. 45).FACS windows were calibrated to ensure that higher affinity clones couldbe discriminated using this approach using HyHEL expressing cells.

The results show a small population of cells that in all cases isclearly separated from the main bulk of non-mutated cells. In cells thathave been newly transfected with the AID expression (panels B and D ofFIG. 45), this population of cells is consistently larger than in thepopulations of cells that did not receive additional AID expressionvector (panels A and C in FIG. 45). These cells were cultured asdescribed below.

Sorted cells were placed in 3 mL DMEM medium containing 10% FBS, 50μg/mL Geneticin, 10 μL/mL Antibiotic-Antimyco tic solution, 1.5 μg/mLpuromycin, 15 μg/mL blasticidin, and 350 μg/mL hygromycin (InvitrogenCorporation, Carlsbad, Calif.) in one well of a 6-well plate. The cellswere cultured until confluent and then archived and reseeded in one wellof a 6-well plate at a cell density of 4×10⁵ cells/mL. The next day, 100μL OptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 4 μL Fugene6(Roche Diagnostics Corporation, Indianapolis, Ind.), and 1 μg of the AIDexpression vector plasmid DNA were mixed and incubated for approximately25-30 minutes at room temperature. After incubation this mixture wasadded drop-wise to the cell culture medium and the cells were culturedand expanded for approximately 7 days. Samples of cells were also takenfor sequence analysis.

Round 2

Cells selected using FITC-HEL in the first round, as described above,were then subjected to the same selection conditions (i.e., incubationwith either 50 or 500 pM FITC-labeled HEL) in a second round of FACSsorting. Fifty milliliters (1e07 cells) of the cells selected from thefirst round were incubated in an ice-cold PBS solution, pH 7.2containing 1% (weight/volume) BSA and either approximately 50 pM or 500pM HEL-FITC for 30 minutes at 4° C. The cell mixture was pelleted, thevolume was reduced to 200 μL and the cells were transferred to sterile-3ml tubes. A 1:500 dilution of PE-conjugated goat-anti-mouseimmunoglobulin was added to the cells and the cells were incubated at 4°C. for 30 minutes. The cells were then pelleted and resuspended 1 mL ofan ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA plus2 nanograms/milliliter DAPI. Live IgG-positive cells that were positivefor FITC (excitation with a 150 mW 488 m laser, collection through a528/38 filter) were isolated by fluorescence activated cell sortingusing a Cytopiea Influx Cell Sorter at a flow rate of approximately10,000 events/second (FIG. 46).

The results of the second sort show a significantly larger population ofcells exhibiting high affinity HEL binding, consistent with theformation of higher affinity mutants by SHM during growth and culture.In cells that have been newly transfected with the AID expressionvector, and then incubated with 500 pM HEL (panel D of FIG. 46) this isclearly a much larger population of highly fluorescent cells, 25.9% ofthe population versus 6.88% compared cells that did not receiveadditional AID expression vector (panel C in FIG. 46). These resultsdemonstrate that re-transformation with the AID expression vector iseffective in promoting a significant improvement in mutagenesis rate.

Continuing this process for 2 additional rounds of mutation withstringent gating on the selected cells (Shown in FIG. 47, panel A)resulted in a profound and significant shift in the binding propertiesof the selected cells (FIG. 47, panel B).

D. Production of Secreted Immunoglobulins for Functional Analysis

Heavy and light chains of interest may be produced in a secreted formfor further functional analysis as described below. In the case of heavychains obtained from the surface displayed libraries, these areprocessed as described in Example 3 (i.e., by digestion with XhoI,followed by re-ligation), to remove the transmembrane domain, enablingdirect secretion of the antibody into the media.

Approximately one day prior to transfection, 3×10⁶ HEK-293 cells wereseeded in 10 mL DMEM/10% FBS medium in a T75 culture flask and incubatedovernight at 37° C. and 5% CO₂. On the day of transfection, 500 μLOptiMEM (Invitrogen Corporation, Carlsbad, Calif.), 20 μL HD-Fugene(Roche Diagnostics Corporation, Indianapolis, Ind.), and 1.5 μg each ofheavy and light chain expression vectors were mixed and incubated forapproximately 25-30 minutes at room temperature. After incubation thismixture was added drop-wise to the cell culture medium.

Approximately three days post-transfection, the cell growth medium wasexchanged with 10 mL Freestyle medium (Invitrogen Corporation, Carlsbad,Calif.) and the cells were incubated for an additional 7 days. At theend of the incubation period, the cell culture supernatants wereharvested and filtered through a sterile 0.2 μm filter. The secretedimmunoglobulins were isolated via standard protein A affinity columnchromatography, prior to BIACORE analysis, as described below.

HEL was immobilized onto a research grade CM5 sensor chip using standardamine coupling. Each of three surfaces was first activated for sevenminutes using a 1:1 mixture of 0.1 mM N-hydroxysuccmimide (NHS) and 0.4mM 1-ethyl-3-(3-dimethylaminopropyl)-carbodimide (EDC). Then, the HELsample is diluted 1- to 50-fold in 10 mM sodium acetate, pH 4.0, andexposed to the activated chip surface for different lengths of time (tenseconds to two minutes) to create three different density surfaces ofHEL. Each surface was then blocked with a seven-minute injection of 1 Methanolamine, pH 8.2. Alternatively biotinylated HEL was diluted100-fold and injected for different amounts of time to be captured atthree different surface densities (60 RU, 45 RU, 12 RU; Response Unit(RU) is termed by Biacore and relates to target molecule per surfacearea) onto a streptavidin-containing sensor chip. All experiments wereperformed on a Biacore® 2000 or T100 optical biosensor. Anti-HELantibodies were supplied at 100 μg/mL and tested in a 3-fold dilutionseries in Sample Running Buffer over HEL conjugated surfaces. Boundanti-HEL antibody was removed using a five-second pulse with sensorregeneration solution. All data was collected at atemperature-controlled 20° C. The kinetic responses for the antibodyinjections were analyzed using the non-linear least squares analysisprogram CLAMP (Myszka, D. G. and Morton, T. A. (1998) Trends Biochem.Sci., 23: 149-150).

E. Sequence Analysis

Sequences of the heavy and light chains isolated in the first sort weredetermined by PCR amplification of heavy and light chains as describedbelow.

At least 50,000 cells taken from populations of interest were pelletedat 1100×g for 5 min. at 4° C. Pelleted cells were resuspended in 15 μLdistilled H₂O and either used immediately in PCR reactions, or werefrozen for later processing.

PCR reactions consisting of 27.6 μL H₂O, 5 μL 10×Pfx buffer, 1 μL cellsfrom above, 8 μL of 2.5 μM of each primer (listed below), and 0.4 μL Pfxpolymerase (Invitrogen Corp., Carlsbad, Calif.) for a total of 50 μLwere run using the following format: 1 cycle of 95° C.×2 min., followedby 35 cycles of 95° C.×30 sec, 55° C. for 30 sec, 68° C. for 45 sec,followed by 1 cycle of 68° C. for 1 min. PCR primers used to amplify theopen reading frames are:

Oligo 540: GTGGGAGGTCTATATAAGCAGAGC (SEQ ID NO: 424), which is a forwardprimer which maps at the 3′ end of a CMV promoter region, approximately140 nucleotides 5′ to the ATG start codon for both heavy and light chainopen reading frames.

Oligo 554: CAGAGGTGCTCTTGGAGGAGGGT (SEQ ID NO: 425), which is a heavychain-specific reverse primer which maps in the IgG gamma chain constantregion.

Oligo 552: ACACAACAGAGGCAGTTCCAGATT (SEQ ID NO: 426), which is a kappalight chain-specific reverse primer that maps near the amino end of thekappa constant region.

Oligo 577: AGTGTGGCCTTGTTGGCTTGAA (SEQ ID NO: 427), which is a lambdalight chain-specific reverse primer that maps to an N-proximal constantregion sequence shared by all five functional human lambda genes (IgL1,2, 3, 6, and 7).

To amplify the heavy chain, oligos 540+554 were used.

To amplify the light chains from a population of cells in which therewas likelihood that a mixture of both kappa and lambda light chainswould be present, oligos 540, 552 and 577 were used simultaneously. Inthis case, the volume of water in the PCR reaction mix was adjusted to19.6 μL.

Following PCR, 5 μL of sample was taken for analysis on an agarose gel.Reactions for which bands were visualized on the gel were then subjectedto further PCR in the presence of Taq polymerase (Invitrogen) using thefollowing conditions:

Added directly to the remaining 45 μL of PCR reaction were 2 μL H₂O, 0.5μL Taq, 0.2 μL dNTPs at 2.5 mM each, and 1.5 μL×50 mM MgCl₂ for a totalof 50 μL (or alternatively, 1 μL of 10× Taq buffer was used in place ofMgCl₂ while adjusting the H₂O to maintain 50 μL final volume). PCRcycling was run as follows: 1 cycle of 95° C.×2 min., followed by 2cycles of 95° C.×30 sec, 55° C. for 30 sec, 72° C. for 45 sec, followedby 1 cycle of 72° C. for 1 min.

Reactions for which bands were either not visualized on the gel or wereotherwise judged to be too weak to continue, were supplemented with 1 μLPfx buffer, 3.7 μL H₂O, and 0.3 μL Pfx polymerase and subjected to 1cycle of 95° C.×2 min., followed by 10 cycles of 95° C.×30 sec, 55° C.for 30 sec, 68° C. for 45 sec, followed by 1 cycle of 68° C. for 1 min.

PCR reactions for which bands were visible following analysis on anagarose gel were cloned using a TOPO® cloning kit from Invitrogenfollowing the manufacturer's suggested protocol. In brief, 4 μL PCRreaction was added to 1 μL salt solution (provided in the TOPO® kit)plus 1 μL TOPO® cloning vector. Following a 20 min. incubation at roomtemp., 1 or 2 μL were used to transform 100 μL XL1 blue as per protocol.

Reading frames from templates whose sequences were of further interestwere recovered as follows: heavy chain templates were recovered bydigesting the TOPO® clones with SgrAI and NheI, which are both presentin all of the original heavy chain sequences. The resultingapproximately 500 bp fragments, which contain the entire variable regionincluding all of CDR3, were cloned into the cognate sites of anexpression vector already comprising the heavy chain constant region togenerate an intact, contiguous heavy chain open reading frame. Oneversion of this vector also contains the transmembrane domain andcytoplasmic tail from the murine H2kk gene as an in-frame fusion withthe IgG1 constant region to permit retention of the final IgG moleculeon the cell surface, as described in Example 3. The alternative versionof the expression vector has the transmembrane deleted to enable directsecretion of the antibodies of interest.

Similarly, light chain templates of interest were removed from theirTOPO® cloning vectors using SbfI and MunI for kappa or SbfI and AclI forlambda, all of which sites are present in the original sequences. Theresulting 350-400 bp fragments, which contain the entire light chainvariable region including CDR3, were cloned into the cognate sites ofthe expression vector to generate an intact, contiguous light chain openreading frame.

The results demonstrated that in approximately 23% of the sequencedclones, there was at least one mutation within the CDR of the lightchain resulting in the mutation of Glycine 31 to Aspartate (G31D). Basedon the crystal structure of HyHEL 10 bound to HEL (Pons et al., (1999)Protein Science 8:958-68), this mutation would be predicted to result inthe formation of an additional hydrogen bonding interaction duringantigen binding, which clearly accounts for the increase in bindingobserved in the presence of 500 pM HEL in FIG. 46, and Biacoremeasurements. Importantly, the type of mutations observed (FIGS. 48A andB) followed the predicted pattern of mutations for SHM mediated mutation(as shown on FIG. 35), and did not result in widespread non-specificmutation of the entire coding regions of the heavy and light chains.These results, therefore, demonstrate the ability of the system toprovide good affinity discrimination, as well as selection of improvedvariants of the antibodies, and binding proteins of the presentinvention, and the ability to provide for both sustained and pulsedhypermutation directed to specific regions of interest within one ormore target proteins. Furthermore, a handful of additional mutationswere identified that, when recombined into a single antibody constructimproved upon the affinity of the wild-type protein, from 30 pM tobetter than 4 PM (FIG. 48C). This example demonstrates how a singlesequence or library under selective pressure and in the presence of SHMcan quickly generate higher affinity mutants, and how this flow ofmutational events can be predicted exactly by the computationalalgorithms outlined above.

The data presented herein demonstrate that the disclosed systems andseed polynucleotides for somatic hypermutation are capable of high leveltargeted mutagenesis of a target protein of interest. Importantly, thesystem is capable of iterative rounds of mutagenesis and selectionenabling the directed evolution of favorable mutations while reducingthe accumulation of neutral and harmful mutations, both within theprotein of interest, and within the expression system.

Example 14 Engineering Enhanced Mutants of AID

Activation induced cytidine deaminase (AID) is the primary enzymeresponsible for initiating somatic hypermutation (SHM), class switchrecombination (CSR) and gene conversion (GC) events during affinitymaturation by the immune system. The enzyme has been especially wellconserved during evolution, with the human, rat, cow, mouse and chickenorthologs exhibiting 94.4%, 93.9%, 93.9%, 92.4% and 89.4% identity tothe canine (dog) amino acid sequence, respectively.

AID contains several predicted protein-protein interaction domains,post-translational modification sites and subcellular targeting motifs,one of which is a nuclear export signal (NES) that is localized in thecarboxy terminal amino acids of the enzyme. The question as to whetheror not a nuclear localization signal (NLS) is present within AID remainscontroversial with some groups claiming such a signal exists (Ito etal., PNAS 2004 Feb. 17; 101(7):1975-80) while others maintain that nofunctional NLS is present (Brar et al., J. Biol. Chem. 2004 Jun. 8;279(25):26395-401; McBride et al., J. Exp. Med. 2004 May 3;199(9):1235-44).

Native AID is found primarily in the cytoplasmic compartment of cells,as demonstrated by cell fractionation, western blotting andimmunohistochemistry. Removal or disabling of the NES tends to permithigher steady-state resident concentrations of AID in the nucleus,higher levels of SHM, but also impaired or absent CSR (Brar et al, Id.;Durandy et al., Hum. Mutat. 2006 December; 27(12):1185-91; Ito et al,Id.; McBride et al, Id.).

Example 2 above describes the design and construction of an SHMresistant form of AID (SEQ ID No. 428) comprising a mutation in the NES(L198A) designed to disable nuclear export thereby promoting nuclearretention. To further enhance nuclear localization and, thus, themutator activity of AID, further engineered versions of the enzyme werecreated by inserting the strong nuclear localization signal (NLS;PKKKRKV; SEQ ID NO: 439) derived from the SV40 T antigen (Kalderon etal, (1984). Cell 39, 499-509) near the amino terminus. To track AIDexpression, a FLAG epitope tag was also inserted to create (SEQ ID No.429) which contains both a strong NLS and the mutant NES sequence.

Additional engineered versions of AID were also created by furthermodifying the C-terminal NES to reduce nuclear export. These constructswere prepared with and without the SV40 T antigen NLS.

In the first pair of NES mutants, polynucleotide sequences of SEQ ID No.428 (without NLS) and SEQ ID No. 429 (with NLS) were modified such thatamino acid residues L181, L183, L189, L196 and L198 encoded by thepolynucleotide sequences were mutated to Alanine resulting inpolynucleotide sequences of SEQ ID No. 431 (without NLS) and SEQ ID No.433 (with NLS), respectively, and amino acid sequences of SEQ. ID. No.432 (without NLS) and SEQ ID No. 434 (with NLS), respectively.

Muteins were generated by PCR, and then treated with Dpn1 to removeparental DNA.

To generate the alanine containing muteins, the following oligos wereused:

(SEQ ID NO: 440) CAGCTCAGGAGAATCCTCGCCCCCGCTTATGAGGTCGACGACCTC and (SEQID NO: 441) GAGGTCGTCGACCTCATAAGCGGGGGCGAGGATTCTCCTGAGCTG.

Two separate PCR reactions were set up using vectors containingpolynucleotide sequences set forth as SEQ ID No. 428 or SEQ ID No. 429as template DNA, using Pfu Taq polymerase (Invitrogen) with themanufacturers kit buffers and 2.5 uM of each deoxynucleotide (Roche).PCR was performed with the following cycle conditions: 1 cycle of 95° C.for 3 min, followed by 20 cycles of [95° C. for 45 sec, 55° C. for 45sec, 68° C. for 17 min], followed by 1 cycle of 68° C. for 5 min. Aftercompletion, 5 μl of the PCR reaction was run on a 1% agarose gel toconfirm a successful reaction. The PCR reaction mix was then treatedwith Dpn1 (New England Biolabs) for at least 4 hrs at 37° C. to removethe parental DNA.

Five (5) μL of the Dpn1-treated PCR reaction was added to 100 μL ofXL1-Blue super competent cells (Invitrogen) and transformed per themanufacturer's suggested protocol. Following sequence verification, theresulting DNA (which contained 2 of the 4 desired mutations; i.e., 181and 183), was used as a template with oligosCCGCTTATGAGGTCGACGACGCCAGAGATGCCTTCCGGACCG (SEQ ID NO: 442) andAGGGTCCGGAAGGCATCTCTGGCGTCGTCGACCTCATAAGCGG (SEQ ID NO: 443) in the sameprotocols listed above to introduce the third of four mutations (i.e.,189). Finally, oligos CCAGAGATGCCTTCCGGACCGCCGGGGCTTGATGTACAATC (SEQ IDNO: 444) and GATTGTACATCAAGCCCCGGCGGTCCGGAAGGCATCTCTGG (SEQ ID NO: 445)were used to incorporate the fourth and final mutation (i.e., 196).

The final set of alanine-containing mutein products were digested usingSac1 and BsrG1 and ligated into vector backbones cut with the cognaterestriction enzymes to generate SEQ. ID. No. 431 (without NLS) and SEQ.ID. No. 433 (with NLS), respectively.

In a second pair of muteins: polynucleotide sequences of SEQ. ID. No.428 (without NLS) and SEQ. ID. No. 429 (with NLS) were modified suchthat amino acid residues Asp187, Asp188 and Asp191 encoded by thepolynucleotide sequences were mutated to Glutamate and amino acidresidue Thr195 encoded by the polynucleotide sequences was mutated toIsoluecine, thereby creating polynucleotide sequences SEQ ID No. 435(without NLS) and SEQ ID No. 437 (with NLS), respectively, and aminoacid sequences of SEQ ID No. 436 (without NLS) and SEQ. ID. No. 438(with NLS), respectively.

The same set of procedures described above with respect to the alaninemuteins was repeated to generate the glutamate containing muteins of AIDSEQ ID No. 435 and SEQ ID No. 437, except that the following oligos:TCCTCCCCCTCTATGAGGTCGAAGAACTCAGAGAAGCCTTCCGGACCCTCGGGGC (SEQ ID NO: 446)and GCCCCGAGGGTCCGGAAGGCTTCTCTGAGTTCTTCGACCTCATAGAGGGGGAGGA (SEQ ID NO:447) were used in place of the first pair of oligos, and the followingoligos: AACTCAGAGAAGCCTTCCGGATCCTCGGGGCTTGATGTACAAT (SEQ ID NO: 448) andATTGTACATCAAGCCCCGAGGATCCGGAAGGCTTCTCTGAGTT (SEQ ID NO: 449) were usedin lieu of the second pair of oligos (no third PCR reaction was neededin this case). Products were treated as described above to generate SEQID No. 435 (without NLS) and SEQ ID No. 437 (with NLS).

Results and Discussion

The six resulting AID constructs were subsequently tested for activityin a green fluorescent protein (GFP) reversion assay, and for frequencyof mutations on an immunoglobulin IgG heavy chain (HC) template.

To perform the GFP reversion assay, the TAC codon for tyrosine 82 wasaltered to a TAG stop codon (GFP*). GFP* was cloned into an Anaptysepisomal expression vector and stably transfected into HEK 293 (note:this cell line expresses EBNA1 from an integrated copy of the gene).Each AID construct in turn was transfected into the stably transfectedGFP* cell line, and cells were placed under selection (blasticidin forGFP* and hygromycin for each of the AID constructs) by day 2 posttransfection. Reversion of the stop codon back to tyrosine caused theepisome-harboring cell to fluoresce green. The frequency of GFPreversion was measured by fluorescence-activated cell sorter (FACS)analysis at 3, 6, and 10 days post selection.

TABLE 16 Table 16 Functional competence of AID muteins as gauged by FACSanalysis of GFP revertant cells gated on days 3, 6, and 10. % gated %gated % gated Vector(s)/AID variants day 3 day 6 day 10 GFP* alone 0.04%0.02% 0.01% GFP* + expression of (SEQ ID No. 428) 0.44% 0.35% 0.39%GFP* + expression of (SEQ ID No. 429) 0.31% 0.37% 0.19% GFP* +expression of (SEQ ID No. 431) 0.19% 0.26% 0.21% GFP* + expression of(SEQ ID No. 433) 0.36% 0.35% 0.32% GFP* + expression of (SEQ ID No. 435)0.37% 0.30% 0.41% GFP* + expression of (SEQ ID No. 437) 0.18% 0.26%0.21%

The results indicate that co-transfection with each of the six AIDconstructs consistently yielded GFP revertants significantly abovebackground, indicating that all 6 muteins of AID are functional.

Because the GFP reversion assay requires both the initial activity ofAID and subsequent action by error prone polymerase in order to generatea positive, reverted cell, the results can provide a qualitative yes/nofor function. In order to determine actual reversion rates, a moreprecise template mutagenesis experiment was also conducted. Thus, inaddition to the GFP reversion assay, 2 of the AID constructs (SEQ ID No.428; containing the L198A mutation in the NES) and SEQ ID No. 429,(containing the L198A NES mutation and the SV40 NLS)) were tested fortheir ability to induce mutations in the HC of HyHEL10 IgG (Pons et al,(1999) Protein Science 8:958-68; Smith-Gill et al. (1984) J. Immunology132:963). Episomal expression constructs (as described previously)encoding the HC of HyHEL10, an N31G mutein of the HyHEL10 light chain(LC), and either an expression vector containing SEQ ID. No. 428 or thesame vector backbone containing SEQ ID. No. 429, were co-transfectedinto HEK 293 cells. Antibiotic selective pressure was added to thetransfected cell population (i.e., blasticidin, puromycin and hygromycinfor HC, LC and AID, respectively), and cells were harvested following 2months of culture. A total of 83 IgG HC templates were sequenced fromcells transfected with an expression vector comprising SEQ ID No. 428,and 61 templates were sequences from cells transfected with anexpression vector comprising SEQ ID No. 429. The percentage of mutationsper template vs. form of AID is shown in Table 17, below. The mutationfrequency calculated from the sequencing data is 1 mutation per 1438 bpgenerated by SEQ ID No. 428, and 1 mutation per 1059 bp generated by SEQID No. 429.

TABLE 17 Table 17 Percentage of HyHEL10 IgG templates identified withmutations observed after co-expression of AID muteins SEQ ID. No. 428 orSEQ ID. No. 430 # Mutations per heavy chain template SEQ ID. No. 428 SEQID. No. 430 0 71%  72% 1 26%  20% 2 2.4%  6.8% 3 0 1.6% 4 0 1.6%

The results indicate that the version of AID that contains the NLS (SEQID No. 429) induced a greater number of mutations in the HyHEL10 HC IgGtemplate (1 per 1059 bp vs 1 per 1438 for the non-NLS containinghomolog), and similarly resulted in a greater number of templatescontaining multiple mutations (10% of templates by AID+NLS vs 2.4% forAID-NLS).

Sequences.

Cold canine AID: nuclear export signal was abrogated by altering theunmodified CTT (Leu198) codon to GCT (ala, shown underlined below).

(SEQ ID NO: 428) ATGGACTCTCTCCTCATGAAGCAGAGAAAGTTTCTCTACCACTTCAAGAACGTCAGATGGGCCAAGGGGAGACATGAGACCTATCTCTGTTACGTCGTCAAGAGGAGAGACTCAGCCACCTCTTTCTCCCTCGACTTTGGGCATCTCCGGAACAAGTCTGGGTGTCATGTCGAACTCCTCTTCCTCCGCTATATCTCAGACTGGGACCTCGACCCCGGGAGATGCTATAGAGTCACTTGGTTTACCTCTTGGTCCCCCTGTTATGACTGCGCCAGACATGTCGCCGACTTCCTCAGGGGGTATCCCAATCTCTCCCTCCGCATATTCGCCGCCCGACTCTATTTTTGTGAGGACAGGAAAGCCGAGCCCGAGGGGCTCAGGAGACTCCACCGGGCCGGGGTCCAGATCGCCATCATGACATTTAAGGACTATTTCTATTGTTGGAATACATTTGTCGAGAATCGGGAGAAGACTTTCAAAGCCTGGGAGGGGCTCCATGAGAACTCTGTCAGACTCTCTAGGCAGCTCAGGAGAATCCTCCTCCCCCTCTATGAGGTCGACGACCTCAGAGATGCCTTCCGGACCCTCGGGGCTTGA

Features of the polynucleotide sequences (or amino acid sequences) arein 5′ to 3′ (or N- to C-terminal where appropriate) as follows:

SacI restriction site used for cloning, boxed letters; Kozak consensus,underlined; ATG start codon (bold capital letters); FLAG epitope tag(single underline); NLS (double-underline); cold canine AID; TGA stopcodon (bold capital letters); BsrGI and AscI restriction sites used forcloning (boxed letters). * indicates stop codon in protein sequence.

Flag-NLS-AID.

The 4 underlined-and-capitalized GCC codons (ala) were changed from theoriginal sequence (CTC encoding Leu) by site directed mutagenesis.

(SEQ ID. No. 431) gagctcctaaccaccATGgactctctcctcatgaagcagagaaagtttctctaccacttcaagaacgtcagatgggccaaggggagacatgagacctatctctgttacgtcgtcaagaggagagactcagccacctctttctccctcgactttgggcatctccggaacaagtctgggtgtcatgtcgaactcctcttcctccgctatatctcagactgggacctcgaccccgggagatgctatagagtcacttggtttacctcttggtccccctgttatgactgcgccagacatgtcgccgacttcctcagggggtatcccaatctctccctccgcatattcgccgcccgactctatttttgtgaggacaggaaagccgagcccgaggggctcaggagactccaccgggccggggtccagatcgccatcatgacatttaaggactatttctattgttggaatacatttgtcgagaatcgggagaagactttcaaagcctgggaggggctccatgagaactctgtcagactctctaggcagctcaggagaatcctcGCCcccGCCtatgaggtcgacgacGCCagagatgccttccggacc GCCggggctTGAtgtaca.(SEQ ID. No. 432) MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILAPAYEVDDARDAFRTAGA*.

The 4 underlined-and-capitalized GCC codons (ala) were changed from theoriginal sequence (CTC encoding Leu) by site directed mutagenesis. Boxesand underlines are as described above.

The 3 underlined-and-capitalized GAA codons (Glu) were changed from theoriginal sequence (Aspartate encoding codons). One additional mutation,T1951, (ACC to ATC) was also generated.

(SEQ ID. No. 435) gagctcctaaccaccATGgactctctcctcatgaagcagagaaagtttctctaccacttcaagaacgtcagatgggccaaggggagacatgagacctatctctgttacgtcgtcaagaggagagactcagccacctctttctccctcgactttgggcatctccggaacaagtctgggtgtcatgtcgaactcctcttcctccgctatatctcagactgggacctcgaccccgggagatgctatagagtcacttggtttacctcttggtccccctgttatgactgcgccagacatgtcgccgacttcctcagggggtatcccaatctctccctccgcatattcgccgcccgactctatttttgtgaggacaggaaagccgagcccgaggggctcaggagactccaccgggccggggtccagatcgccatcatgacatttaaggactatttctattgttggaatacatttgtcgagaatcgggagaagactttcaaagcctgggaggggctccatgagaactctgtcagactctctaggcagctcaggagaatcctcctccccctctatgaggtcGAAGAActcagaGAAgccttccggATC ctcggggctTGAtgtaca(SEQ ID. No. 436) MDSLLMKQRKFLYHFKNVRWAKGRHETYLCYVVKRRDSATSFSLDFGHLRNKSGCHVELLFLRYISDWDLDPGRCYRVTWFTSWSPCYDCARHVADFLRGYPNLSLRIFAARLYFCEDRKAEPEGLRRLHRAGVQIAIMTFKDYFYCWNTFVENREKTFKAWEGLHENSVRLSRQLRRILLPLYEVEELREAFRILGA*

The 3 underlined-and-capitalized GAA codons (Glu) were changed from theoriginal sequence (Aspartate encoding codons). One additional mutation,T195I (ACC to ATC) was also generated. Boxes and underlines are asdescribed above.

Example 15 Discovery and Optimization of NGF Antibodies

Nerve growth factor (NGF) has been shown to be a critical survival andmaintenance factor in the development of peripheral sympathetic andembryonic sensory neurons and of basal forebrain cholinergic neurons(Smeyne, et al., Nature 368:246-249 (1994); Crowley, et al., Cell76:1001-1011 (1994)).

NGF activity is mediated through two different membrane-bound receptors,the TrkA tyrosine kinase receptor and the p75 receptor, which arestructurally related to other members of the tumor necrosis factorreceptor family (Chao, et al., Science 232:518-521 (1986)). NGFreceptors have been found on a variety of cell types outside of thenervous system. For example, TrkA has been found on human monocytes, T-and B-lymphocytes and mast cells.

A direct relationship between increased NGF levels and a variety ofinflammatory conditions has been established in human patients as wellas in several animal models. These include systemic lupus erythematosus(Bracci-Laudiero, et al., Neuroreport 4:563-565 (1993)), multiplesclerosis (Bracci-Laudiero, et al., Neurosci. Lett. 147:9-12 (1992)),psoriasis (Raychaudhuri, et al., Acta Derm l'enereol. 78:84-86 (1998)),arthritis (Falcimi, et al., Ann. Rheuim Dis. 55:745-748 (1996)),interstitial cystitis (Okragly, et al., J. Urology 161:438-441 (1991))and asthma (Braun, et al., Eur. J. Immunol. 28:3240-3251 (1998)).

Primary sympathetic neurons are also known to respond to NGF and to alsobe involved in pain signaling (Aley, et al., Neuroscience 71:1083-1090(1996)). Removing sympathetic innervation modifies the hyperalgesianormally seen in response to treatment with NGF (Woolf, et al., J.Neurosci. 16:2716-2723 (1996)).

The use of anti-NGF antibody to treat chronic pain has been describedUnited States Patent Application Nos. 20040219144, 20040228862,20040237124, 20040253244, 20050074821, 20050265994, 20060088884 and20060147450.

1. Generation of Reagents

Preparation Of Cell Surface Expressed Libraries

The preparation and cell banking of a HEK-293 cell line expressing alibrary of membrane-bound human antibody genes is described in Examples5-8. This cell line also comprises an AID expression vector as describedin Example 3, which is capable of constitutive AID expression. A HEK-293cell line expressing AID, but not the antibody library was used as anegative control for the selections.

2. Selection of Specific Binding Members

For the first two rounds of selection using intact human NGF protein,200 microliters of cells were incubated with approximately 1×10⁷fully-saturated human NGF-conjugated Spherotech avidin purple(Spherotech, Lake Forest, Ill.) beads for 30 minutes at 4° C.

Prior to incubation with NGF bound beads, the cells were collected,washed with an equal volume of PBS solution, pH 7.2 and resuspended inice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSA at afinal cell concentration of 5×10⁷ cells/mL.

NGF-conjugated beads were prepared by incubation of the biotinylatedprotein with the streptavidin beads for 30 minutes at room temperaturewith slow tilt rotation as described previously. After coupling, themicroparticles were washed and resuspended to a final microparticleconcentration of 1× O₉ microparticles/ml. Prior to coupling to beads,NGF was biotinylated usingsulfosuccinimidyl-6-(biotinamido)-6-hexanamidohexanoate (biotin reagent;Pierce product number 21338, Pierce, Rockford, Ill. 61105).

After incubation, the cell: bead mixture was washed once with ice-coldPBS solution, pH 7.2 containing 1% (weight/volume) BSA and resuspendedin 200 microliters of ice-cold PBS solution, pH 7.2 containing 1%(weight/volume) BSA containing a 1:200 dilution of anti-IgG antibodies,as described previously. The cells were incubated at 4° C. for 30minutes and then washed once with ice-cold PBS solution, pH 7.2containing 1% (weight/volume) BSA and resuspended in 500 microliters ofsterile ice-cold PBS solution, pH 7.2 containing 1% (weight/volume) BSAplus 2 nanograms/milliliter DAPI (Sigma-Aldrich Corp). Live IgG-positivecells that bound NGF-conjugated beads (excitation with a 25 mW 561 nmlaser, collection through 620/40 and 750LP filters, respectively) wereisolated by fluorescence activated cell sorting using a Cytopiea InfluxCell Sorter at a flow rate of approximately 10,000 events/second. In thefirst round of selection, the entire population of cells which bound tohuman NGF were isolated plated in a 6-well microtiter dish and allowedto expand for approximately 2 weeks to a population of approximately1×10⁷ cells before the next sort FIG. 49 (Panel A). The second round ofsorting resulted in a significantly enriched population of NGF bindingcells, and the most fluorescent cells were taken and allowed to expandas described above (Panel B of FIG. 49). Controls for these experimentsare shown in the smaller insert panels. Samples of the cells from round2 can be processed to determine the sequences of the variable domainsand to characterize diversity of selected heavy and light chains, asdescribed previously.

For the third round of selections with NGF protein, 200 microliters ofthe selected cells that bound to NGF from round 2 were incubated withapproximately 50 nmolar biotinylated human NGF. The cell mixture waswashed once with ice-cold PBS solution, pH 7.2 containing 1%(weight/volume) BSA followed by incubation with a 1:200 dilution of 1mg/mL PE-conjugated streptavidin at 4° C. for 30 minutes. The cells werewashed once with ice-cold PBS solution, pH 7.2 containing 1%(weight/volume) BSA and incubated with goat anti human IgG-FITC(Sigma-Aldrich Corp., St. Louis, Mo.) at a 1:500 dilution (2 μL).Samples were vortexed and incubated 4° C. for 30 minutes. The cells werethen washed once with ice-cold PBS solution, pH 7.2 containing 1%(weight/volume) BSA and resuspended in 500 microliters of sterileice-cold PBS solution, pH 7.2, containing 1% (weight/volume) BSA plus 2nanograms/milliliter DAPI (Sigma-Aldrich Corp). Live IgG-positive cellsthat were positive for phycoerythrin (excitation with the 25 mW 561 mlaser, collection through 579/34) were isolated by fluorescenceactivated cell sorting using a Cytopeia Influx Cell Sorter at a flowrate of approximately 10,000 events/second. Subpopulations of cells thatbound to human NGF are shown in FIG. 49 (Panel C) Sorted cells with thehighest fluorescence intensity were plated in a 6-well microtiter dishto expand for approximately 2 weeks to a population of approximately1×10⁷ cells before the next sort.

For the fourth round of selection, cells were selected using 20 nMbiotinylated human NGF, using the same procedures and incubations, asdescribed above. The results of the FACS sorts are shown in FIG. 49(Panel D).

For the fifth round of selection, cells were selected using 20 mMbiotinylated human NGF using the same FACS procedures and incubations,as described above. The results of the FACS are shown in FIG. 49 (PanelE) and the results demonstrate a significant enrichment in bindingcompared to control incubations (insert panels in FIG. 49). In Panel Eof FIG. 49, it is clear that selections using intact NGF protein show afinger of cells in the FACS scattergram exhibiting discrete binding.Selected subpopulations of cells were plated in a 6-well microtiter dishto expand for approximately 2 weeks to a population of approximately1×10⁷ cells before the next sort.

Samples of the cells from round 5 were processed to determine thesequences of the variable domains compared to round 2 results. Asdiscussed previously, the sequences of the variable domains and newmutations introduced into the antibody via somatic hypermutation can beanalyzed to determine their distribution within the clones analyzed, andspecifically their location within the coding region of the heavy andlight chains. Mutations can be rated based on their position within theheavy light chains; for example mutations within the CDRs can be ratedhighly, while those in frameworks regions and/or the constant domainscan be rated less favorably. Key mutations that occur between differentantibody families may be recombined between families to rapidly generatehybrid antibodies that exhibit favorable increases in affinity orselectivity that represent the sum of all, or a sub set of all,mutations observed. Conversely, multiply redundant clonal families canbe consolidated to eliminate redundant diversity while maximizing usefuldiversity and eliminating non productive evolutionary paths.

3. Clonal Analysis

For each cell clone, the sequencing template is prepared either via PCRor episomal rescue, as described above in Example 13.

4. Functional Analysis

Heavy and light chains of interest after sequence analysis may beproduced in a secreted form for further functional analysis as describedbelow. In the case of heavy chains obtained from the surface displayedlibraries, these are processed as described in Example 13, (i.e., bydigestion with XhoI, followed by religation) to remove the transmembranedomain, enabling direct secretion of the antibody in to the media.Purified antibodies may be tested in any of the functional assays belowto further characterize antibody activity.

A number of assays can be developed to help reveal prospectivefunctional activity of isolated antibodies or within antibody pools asdescribed below:

5. Affinity Analysis

A heavy and light chain pair of interest after sequence analysis fromRound 5 were produced in a secreted form for further functional analysisas described above. The conditioned media containing the antibody inquestion was purified by Protein G affinity chromatography, and dialyzedinto running buffer appropriate for the Biacore affinity experiments,typically phosphate saline buffer (PBS) pH 7.4.

Affinity analysis of the Round 5 anti-NGF lead antibody was performed ona Biacore series T-100 surface plasmon resonance instrument with thefollowing experimental conditions. A CM5 Biacore chip was conditionedwith Protein G to create a broad-spectrum anti-human IgG capturesurface. A series of NGF ligand concentrations were passed over the chipsurface on which either 500RU of anti-NGF antibody had been captured, orno anti-NGF antibody had been captured (control surface). The rate ofconcentration-dependent association and dissociation of the analyte,NGF, was monitored as a function of time on the capture cell relative tothe control flow cell. Results of these experiments can be seen in FIG.50, which demonstrate a concentration-dependent association anddissociation of NGF to the antibody in question.

A kinetic multivariate analysis of these binding data shown in FIG. 50predict a dissociation constant for binding of NGF to the anti-NGFantibody of K_(d)=670 nM. The off-rate (k_(d)) is predicted to be 0.367(s⁻¹), with an association rate of k_(on)=5.5×10⁵ (s⁻M⁻¹). These datademonstrate that we have isolated an anti-NGF antibody that binds to NGFwith nM affinity using the protocols described above.

Example 16 Creation and Testing of Synthetic SHM Resistant and SHMSusceptible Genes

A. Polynucleotide Design

The starting sequence for unmodified Teal Fluorescent Protein (TFP) isshown in FIG. 51, together with the initial analysis of hot spot andcold spot frequency.

1. Hot TFP

As described for Example 1, sequence optimization is completed using thecomputer program SHMredesign, based on the hot spot and cold spot motifslisted in Table 7; the resulting hot and cold versions of TFP are shownin FIGS. 52 and 53, respectively.

Optimization of the TFP sequence to make the sequence more susceptibleto somatic hypermutation resulted in an increase of about 170% in numberof hot spots (an increase of 28), and reduced the number of cold spotsby about 26% (a decrease of 27). Overall the frequency of hot spotsincreased to an average density of about 10 hot spots per 100nucleotides from an initial density of about 6 hot spots per 100nucleotides, and the overall frequency of cold spots decreased fromabout 15 cold spots per 100 nucleotides in the unmodified gene to about11 cold spots per 100 nucleotides in the SHM susceptible form.

2. Cold TFP

Optimization of the TFP sequence to make the sequence more resistant tosomatic hypermutation resulted in an increase of 120% in number of coldspots (an increase of 21), and reduced the number of hot spots by about10% (a decrease of 4). Overall the frequency of cold spots increased toan average density of about 18 cold spots per 100 nucleotides from aninitial density of about 15 cold spots per 100 nucleotides, and theoverall frequency of hot spots decreased from about 6 hot spots per 100nucleotides, in the unmodified gene to about. 5 hot spots per 100nucleotides in the SHM resistant form.

B. Cloning and Analysis

After final review to ensure that the synthetic polynucleotide sequenceis free of extraneous restriction sites, the complete polynucleotidesequence is synthesized (DNA 2.0, Menlo Park, Calif.), cloned into oneof DNA2.0's cloning vectors as describe herein, sequenced to confirmcorrect synthesis and tested for activity as described below.

Hek 293 cells are transfected with the expression vectors (AB102 and 136as described above) containing either hot or cold versions of TFP drivenfor expression by an identical CMV promoter. Selection for stableexpression began 3 days post transfection. Prior to FACS analysis, cellsare harvested by trypsinization, ished twice in PBS containing 1% w/vBSA, and re-suspended in 200 μl PBS/i % BSA containing 2 ng/ml DAPI.Cells are analyzed in the Cytopeia Influx with 200 mW 488 nm and 50 mW403 nm laser excitation. Up to one million cells per sample areacquired. DAPI fluorescence is measured through a 460/50 bandpassfilter. GFP fluorescence is measured through a 528/38 bandpass filter.Percent GFP expression is reported in Table 18 as percentage of DAPIexcluding live cells with no detectable GFP fluorescence above cellularbackground.

TABLE 18 Expression analysis of “hot” and “cold” versions of TFP % TFPExpressing TFP Control Fold over Construct cells FluorescenceFluorescence control Hot TFP (SHM 63.74 189.33 20.61 9 susceptible) ColdTFP (SHM 66.92 429.72 19.93 22 resistant) Hot TFP (SHM 48.39 183.2120.09 9 susceptible) Cold TFP (SHM 51.20 656.06 20.26 32 resistant)

These results show good expression above background of both hot and coldversions of TFP. In this case, making the sequence “cold” produced thesurprising result that relative expression of the protein is improved.Such improved expression provides an additional benefit to the SHMresistant synthetic genes.

To determine the relative stability/susceptibility of each construct tosomatic hypermutation, stable cell lines of each transfected cellpopulation are created, and tested to determine the relative speed bywhich they accumulate SHM mediated mutations. Because the majority ofthese mutations result in a loss of function, relative mutagenesis loadare conveniently measured as a loss of fluorescence via FACS asdescribed herein.

Episomal expression constructs carrying either a SHM optimized codingsequence for hot TFP or cold TFP were individually stably co-transfectedwith AID into HEK 293 cells and allowed to expand and grow for 3 weeks(the cold canine AID used in these experiments contains theNES-inactivating L198A mutation; SEQ ID NO: 428). Cell stocks were thenfrozen, and one vial each of hot TFP and cold TPF were thawed, grown inculture for 4 days, and then pulsed with supplemental AID by transientlytransfecting the 4 day post-thaw culturing with an additional aliquot ofthe original AID expression construct (termed “AID pulsing”). Cells wereharvested by trypsinization nine days following the AID pulse, pelletedat 1150×g for 5 min., and frozen for later use.

Cell pellets were subsequently thawed and TFP ORFs were recovered by PCRusing oligonucleotide (oligo) primers

GTGGGAGGTCTATATAAGCAGAGC (SEQ ID NO: 456) and GATCGTCTCACGCGGATTGTAC.(SEQ ID NO: 457)

The former oligo amplifies from near the 3′ end of the CMV promoter usedfor driving expression of TFP mRNA, which lies 142 nt 5′ to the TFPstart codon, and the latter oligo matches sequences ending 1 nt 3′ tothe TFP stop codon. Each PCR reaction (total volume of 50 μL) was run 35cycles under the following conditions: 95° C. for 5 min, 35 cycles of(95° C. for 30 sec, 55° C. for 30 sec, 68° C. for 45 sec), followed by 1min at 68° C. before cooling to 4° C. PCR amplified products were clonedinto the TOPO® TA cloning vector (Invitrogen, Carlsbad, Calif.), andinserts were sequenced. A total of 166 hot and 111 cold TFP ORFs wererescued, sequenced and compared the resulting spectrum of mutations.Global statistics for the mutations observed in the two sets ofsequences are shown in Table 19.

TABLE 19 Mutation metrics for cold- and hot-TFP # ORFs # total # nt kbper templates per template sequenced mutations sequenced mutationmutation coldTFP 111 18 61050 3391 6.1 hotTFP 166 100 88500 885 1.6

The mutation frequency is approximately 3.8-fold greater in the TFPtemplate version with maximized hotspots vs. the cold TFP sequence withminimized hotspots. The data demonstrates that SHM optimization ofpolynucleotide sequences can be used to either increase or decrease thefrequency of mutations experienced by a polynucleotide encoding aprotein of interest.

FIG. 53D shows the mutations for a representative segment of the hot andcold TFP constructs. The central row shows the amino acid sequence ofTFP (residues 59 thru 87) in single letter format, and the “hot” and“cold” starting nucleic acid sequences encoding the two constructs areshown above (hot) and below (cold) the amino acid sequence. Mutationsobserved in the hot sequence are aligned and stacked top of the genesequences, while mutations in the cold TFP sequence are shown below. Theresults illustrate how “silent” changes to the coding sequences generatedramatic changes in observed AID-mediated SHM rates, demonstrating thatengineered sequences can be effectively optimized to create fast or slowrates of SHM.

FIG. 53E shows that the spectrum of mutations generated by AID in thepresent in vitro tissue culture system mirror those observed in otherstudies and those seen during in vivo affinity maturation. FIG. 53Eshows the mutations generated in the present study (Box (i) upper left,n=118), and compares them with mutations observed by Zan et al. (box(ii) upper right, n=702), Wilson et al. (lower left, n=25000; box(iii)), and a larger analysis of IGHV chains that have undergoneaffinity maturation (lower right, n=101,926; box (iv)). The Y-axis ineach chart indicates the starting nucleotide, the X-axis indicates theend nucleotide, and the number in each square indicates the percentage(%) of time that nucleotide transition is observed. In the presentstudy, the frequency of mutation transitions and transversions wassimilar to those seen in other data sets. Mutations of C to T and G to Aare the direct result of AID activity on cytidines and account for 48%of all mutation events. In addition, mutations at bases A and T accountfor ˜30% of mutation events (i.e., slightly less than frequenciesobserved in other datasets).

FIG. 53F shows that mutation events are distributed throughout the SHMoptimized nucleotide sequence of the hot TFP gene, with a maximuminstantaneous rate of about 0.08 events per 1000 nucleotides pergeneration centered around 300 nucleotides from the beginning of theopen reading frame. Stable transfection and selection of a gene with AID(for 30 days) produces a maximum rate of mutation of 1 event per 480nucleotides. As a result, genes may contain zero, one, two or moremutations per gene. The distribution of SHM-mediated events observed inhot TFP sequenced genes can be seen in FIG. 53G, compared to thesignificantly reduced pattern of mutations seen in cold TFP (FIG. 53H).

Thus the present study demonstrates that the creation of non-synonymousversions of genes such as Teal-fluorescent protein (TFP) that do notnormally undergo somatic hypermutation can be used to target such genesfor high rates of somatic hypermutation. Additionally, the creation ofSHM resistant genes (while encoding for the same amino acids) can leadto proteins that have a reduced number of somatic hypermutationhot-spots and, thus, experience a dramatically reduced level of AIDmediated hypermutation. In each instance of SHM optimization, mammaliancodon usage and other factors effecting gene expression levels wereconsidered in generating the engineered sequences, leading to proteinsthat also exhibit reasonable levels of translation and expression. Theresults, therefore, demonstrate that the present methods of SHMoptimization (i) can be successfully used to target the activity of AIDto specific regions of an expressed gene; (ii) can be used to speed orslow the rate of SHM, (iii) demonstrate that the spectrum of mutationsgenerated by AID using this methodology is equivalent to that observedin vivo; (iv) and demonstrate that SHM optimization can be successfullyperformed on a gene of interest to either positively or negativelyimpact its rate of AID-mediated SHM without significantly negativelyimpacting its expression.

While preferred embodiments of the present invention have been shown anddescribed herein, such embodiments are provided by way of example only.It should be understood that various alternatives to the embodiments ofthe invention described herein may be employed in practicing theinvention. It is intended that the following claims define the scope ofthe invention and that methods and structures within the scope of theseclaims and their equivalents be covered thereby.

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1. A method of making a protein of interest with a desired property,said method comprising the steps of: a. synthesizing a seed library ofpolynucleotides encoding a plurality of one or more polypeptide speciesof interest that have at least one region of interest of a protein ofinterest, wherein said seed library of polynucleotides comprise at leastone synthetic nucleic acid sequence that encodes said at least oneregion of interest which has been optimized for SHM; b. joining inoperable combination said seed library of polynucleotides encoding saidplurality of one or more polypeptide species of interest of a protein ofinterest into an expression vector; c. transforming a host cell withsaid expression vector, so that said protein of interest is produced byexpression of said seed library of polynucleotides encoding a pluralityof one or more polypeptide species of interest of a protein of interest;and wherein said host cell expresses AID activity or can be induced toexpress AID activity via the addition of an inducing agent; d.optionally inducing AID activity, or allowing AID mediated mutagenesisto occur on said seed library of polynucleotides; e. identifying a cellor cells within the population of cells which expresses a mutatedprotein having said desired property, and f. establishing one or moreclonal populations of cells from the cell or cells identified in step(e).
 2. The method of claim 1, wherein at least one said syntheticnucleic acid sequence has been optimized for SHM by reducing the densityof non-preferred codons.
 3. The method of claim 1, wherein at least onesaid synthetic nucleic acid sequence has been optimized for SHM by theinsertion of one or more WAC motifs, one or more WRC motifs, or acombination thereof.
 4. The method of claim 1, wherein at least one saidsynthetic nucleic acid sequence has been optimized for SHM by theinsertion of one or more preferred SHM codons.
 5. The method of claim 1,wherein said insertion of one or more preferred SHM codons is byinsertion of one or more amino acids substitutions in said region ofinterest, said amino acid substitutions being silent, conservative,semi-conservative, non-conservative or a combination thereof.
 6. Themethod of claim 1, wherein at least one said synthetic nucleic acidsequence has been optimized for SHM by increasing the density ofcanonical hot spots.
 7. The method of claim 1, wherein said host cell isa prokaryotic cell or a eukaryotic cell.
 8. The method of claim 1,wherein said seed library of polynucleotides further comprise at leastone polynucleotide sequence that has been made resistant to SHM.
 9. Amethod of making an antibody or antigen-binding fragment thereof with adesired property, said method comprising the steps of: a. synthesizing afirst seed library of first polynucleotides encoding a plurality of oneor more antibody heavy chain proteins or fragments that have at leastone heavy chain CDR, wherein said first seed library of polynucleotidescomprise at least one first synthetic nucleic acid sequence that encodessaid at least one heavy chain CDR which has been optimized for SHM; b.synthesizing a second seed library of second polynucleotides encodingsaid plurality of one or more antibody light chain proteins or fragmentsthat have at least one light chain CDR, wherein said second seed libraryof polynucleotides comprise at least one second synthetic nucleic acidsequence that encodes said at least one light chain CDR and has beenoptimized for SHM; c. joining in operable combination said first andsecond seed libraries of polynucleotides encoding said plurality ofantibody heavy chain proteins or fragments thereof and said seed libraryof polynucleotides encoding said plurality of antibody light chainproteins or fragments thereof into expression vectors; d. transforming ahost cell with said expression vectors, so that an antibody or anantigen-binding fragment thereof is produced by coexpression of a heavychain sequence from said seed library of polynucleotides encoding aplurality of antibody heavy chain proteins or fragments thereof and alight chain sequence from said seed library of polynucleotides encodinga plurality of antibody light chain proteins or fragments thereof,either on the same or different expression vectors; and wherein saidhost cell expresses AID activity or can be induced to express AIDactivity via the addition of an inducing agent; e. optionally inducingAID activity, or allowing AID mediated mutagenesis to occur on said seedlibraries of polynucleotides; f. identifying a cell or cells within thepopulation of cells which expresses a mutated antibody or anantigen-binding fragment thereof having the desired property, and g.establishing one or more clonal populations of cells from the cell orcells identified in step (f).
 10. The method of claim 9, wherein atleast one said first or second synthetic nucleic acid sequences havebeen optimized for SHM by reducing the density of non-preferred codons.11. The method of claim 9, wherein at least one said first or secondsynthetic nucleic acid sequences, have been optimized for SHM by theinsertion of one or more WAC motifs, one or more WRC motifs, or acombination thereof.
 12. The method of claim 9, wherein at least onesaid first or second synthetic nucleic acid sequences have beenoptimized for SHM by the insertion of one or more preferred SHM codons.13. The method of claim 12, wherein said insertion of one or morepreferred SHM codons is by insertion of one or more amino acidssubstitutions in said at least one heavy chain or light chain CDR, saidamino acid substitutions being silent, conservative, semi-conservative,non-conservative or a combination thereof.
 14. The method of claim 9,wherein said host cell is a prokaryotic cell or a eukaryotic cell. 15.The method of claim 14, wherein said prokaryotic cell is an E. colicell.
 16. The method of claim 15, wherein said eukaryotic cell is amammalian cell or a yeast cell.
 17. The method of claim 9, wherein saidfirst or second seed library of polynucleotides further comprise atleast one polynucleotide sequence that has been made resistant to SHM.18. A method of co-evolving a plurality of proteins, said methodcomprising the steps of: a. synthesizing a first seed library ofpolynucleotides encoding a plurality of one or more polypeptide speciesof interest that have at least one region of interest of a first proteinof interest, wherein said first seed library of polynucleotides compriseat least one first synthetic nucleic acid sequence that encodes said atleast one first region of interest which has been optimized for SHM; b.synthesizing a second seed library of polynucleotides encoding aplurality of one or more polypeptide species of interest that have atleast one region of interest of a second protein of interest, whereinsaid second seed library of polynucleotides comprise at least one secondsynthetic nucleic acid sequence that encodes said at least one secondregion of interest and has been modified to act as a substrate for AIDmediated somatic hypermutation; c. joining in operable combination saidfirst and second seed libraries of polynucleotides encoding saidplurality of polypeptide species of interest of said first protein ofinterest and said seed library of polynucleotides encoding saidplurality of polypeptide species of interest of said second protein ofinterest into expression vectors; d. transforming a host cell with saidexpression vectors, so that said first and second proteins of interestare produced by coexpression of said first and second seed libraries ofpolynucleotides, either on the same or different expression vectors; andwherein said host cell expresses AID activity, or can be induced toexpress AID activity via the addition of an inducing agent; e.optionally inducing AID activity, or allowing AID mediated mutagenesisto occur on said seed libraries of polynucleotides; f. identifying acell or cells within the population of cells which expresses a mutatedfirst or second protein of interest having the desired property, and g.establishing one or more clonal populations of cells from the cell orcells identified in step (f).
 19. The method of claim 18, wherein atleast one said first or second synthetic nucleic acid sequences havebeen optimized for SHM by reducing the density of non-preferred codons.20. The method of claim 18, wherein at least one said first or secondsynthetic nucleic acid sequences, have been optimized for SHM by theinsertion of one or more WAC motifs, one or more WRC motifs, or acombination thereof.
 21. The method of claim 18, wherein at least onesaid first or second synthetic nucleic acid sequences have beenoptimized for SHM by the insertion of one or more preferred SHM codons.22. The method of claim 21, wherein said insertion of one or morepreferred SHM codons is by insertion of one or more amino acidssubstitutions in said first or second region of interest, said aminoacid substitutions being silent, conservative, semi-conservative,non-conservative or a combination thereof.
 23. The method of claim 18,wherein said host cell is a prokaryotic cell or a eukaryotic cell. 24.The method of claim 23, wherein said prokaryotic cell is an E. colicell.
 25. The method of claim 23, wherein said eukaryotic cell is amammalian cell or a yeast cell.
 26. The method of claim 18, wherein saidfirst or second seed libraries of polynucleotides further comprise atleast one polynucleotide sequence that has been made resistant to SHM.27. A composition of matter comprising a seed library of polynucleotidesencoding a plurality of one or more polypeptide species of interest thathave at least one region of interest of a protein of interest, whereinsaid seed library of polynucleotides comprise at least one syntheticnucleic acid sequence that encodes said at least one region of interestwhich has been optimized for SHM by the insertion of one or morepreferred SHM codons.
 28. The composition of claim 27, wherein saidsynthetic nucleic acid sequence has been optimized for SHM by reducingthe density of non-preferred codons.
 29. The composition of claim 27,wherein said insertion of one or more preferred SHM codons is byinsertion of one or more amino acids substitutions into said region ofinterest, said amino acid substitutions being silent, conservative,semi-conservative, non-conservative or a combination thereof.
 30. Thecomposition of claim 27, wherein said synthetic nucleic acid sequencehas been optimized for SHM by the insertion of one or more WAC motifs,one or more WRC motifs, or a combination thereof.
 31. The composition ofclaim 27, wherein said seed library of polynucleotides encodes anantibody or antigen-binding fragment thereof.
 32. The composition ofclaim 27, wherein said seed library of polynucleotides encodes anantibody heavy chain or fragment thereof.
 33. The composition of claim27, wherein said seed library of polynucleotides encodes an antibodylight chain or fragment thereof.
 34. The composition of claim 32,wherein said antibody heavy chain comprises a variable region selectedfrom among the antibody heavy chains of FIG. 20A.
 35. The composition ofclaim 34, wherein said antibody heavy chain comprises a variable regionselected from the group consisting of IGHV6-1, IGHV4-34, IGHV4-59,IGHV3-30-3, IGHV3-7, IGHV3-23, IGHV5-51, IGHV1-2 and IGHV1-69.
 36. Thecomposition of claim 33, wherein said antibody light chain comprises avariable region selected from among the antibody light chains of FIG.20B.
 37. The composition of claim 36, wherein said antibody light chaincomprises a κ light chain variable region selected from the groupconsisting of IGKV2D-30, IGKV4-1, IGKV1-33, IGKV1D-39 and IGKV3-20. 38.The composition of claim 33, wherein said antibody light chain comprisesa λ variable region selected from among the antibody light chains ofFIG. 20C.
 39. The composition of claim 38, wherein said antibody lightchain comprises a λ light chain variable region selected from the groupconsisting of IGKLV743, IGLV1-40, IGLV2-11 and IGLV3-21.
 40. Thecomposition of claim 27, wherein said at least one region of interestcomprises an antibody heavy or light chain CDR1, CDR2 or CDR3 domain.41. The composition of claim 27, wherein said at least one region ofinterest comprises an antibody heavy or light chain CDR3.
 42. Thecomposition of claim 27, wherein said protein of interest is a receptor,an enzyme, a co-factor or a transcription factor.
 43. The composition ofclaim 27, wherein said seed library of polynucleotides further containsat least one polynucleotide sequence that has been made resistant toSHM.